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USER GUIDE

DNA Fragment Analysis


by Capillary Electrophoresis

Publication Number 4474504


Revision B

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For Research Use Only. Not intended for use in diagnostic


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For Research Use Only. Not intended for use in diagnostic procedures.
The information in this guide is subject to change without notice.
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UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN
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TRADEMARKS
All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.
AmpErase, AmpliTaq, AmpliTaq Gold, and TaqMan are registered trademarks of Roche Molecular Systems, Inc.
AFLP is a registered trademark of Keygene N.V.
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2014 Thermo Fisher Scientific Inc. All rights reserved.

Contents

About This Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13


Revision history . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Purpose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Prerequisites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Structure of this guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

CHAPTER 1

Introduction to Fragment Analysis . . . . . . . . . . . . . . . . . . 15

Fragment analysis versus sequencingwhat is the difference? . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15


Fragment analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
What can I do with fragment analysis? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Types of applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Applications described in this guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
What is capillary electrophoresis? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Fragment analysis workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

CHAPTER 2

Experimental Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

Experimental design considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21


DNA polymerase enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Derivatives of Tth DNA polymerase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Enzyme characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Fluorescent labeling methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25


Singleplexing versus multiplexing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Singleplexing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Multiplexing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Multiplexing (pooling) strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Multiplex design software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Multiplexing guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Primer design guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .


Primer design criteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Primer design software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Factors affecting Tm and primer annealing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Effects of template secondary structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Selective amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Preferential amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Contents

Non-specific amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Minimizing binding to other primers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Post-amplification manipulations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Addition of 3' A nucleotide by Taq polymerase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Dyes and chemical forms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Multicomponent analysis with fluorescent dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Factors that affect dye signal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Emission and absorption (excitation) wavelengths and relative intensities . . . . . . . . . . . . . . .
Points to consider when selecting dyes for custom primers . . . . . . . . . . . . . . . . . . . . . . . . . . .
Example: selecting dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Dye sets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Dye sets and matrix standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Creating a custom dye set . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Size standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Functions of a size standard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Size-standard peak intensity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Selecting a GeneScan size standard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Peaks not used for sizing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Preparing a size standard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
GeneScan 120 LIZ Size Standard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
GeneScan 500 LIZ Size Standard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
GeneScan 600 LIZ and GeneScan 600 LIZ v2.0 Size Standards . . . . . . . . . . . . . . . . . . . .
GeneScan 1200 LIZ Size Standard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
GeneScan 350 ROX Size Standard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
GeneScan 400HD ROX Size Standard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
GeneScan 500 ROX Size Standard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
GeneScan 1000 ROX Size Standard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Ordering custom primers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52


Testing the primers and optimizing conditions with test DNA panel . . . . . . . . . . . . . . . . . . . . . . . . . . 52
Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
Optimizing conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

CHAPTER 3

Optimizing PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

Safety information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

Isolating, purifying, quantifying, and storing DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .


Isolating DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Purifying DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Quantifying DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Storing prepared DNA before or after PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Handling primers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Reconstituting and diluting primers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Quantifying primers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Storing primers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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DNA Fragment Analysis by Capillary Electrophoresis

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Using control DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .


Purpose of control DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Guidelines for use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
CEPH 1347-02 Control DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Reaction volumes and plate types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .


Reaction volumes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Using small amounts of template . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Plate types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Reagent concentrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
dNTP concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Magnesium ion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Template concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Enzyme concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Preventing competing side reactions: hot-start PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60


When to use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
Limitations and alternatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
Thermal cycling parameters Veriti Thermal Cyclers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
AmpliTaq_Gold . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
General PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Time-release PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Touchdown PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Thermal cycling parameters 9700 Thermal Cyclers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .


AmpliTaq_Gold . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
General PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
LMS2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Time Release PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Touchdown PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
XL PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Optimizing thermal cycling parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63


Optimizing temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
Avoiding contamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
PCR setup work area . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Amplified DNA work area . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Avoiding contamination from the environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Avoiding PCR product carryover . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
For more information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

CHAPTER 4

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Optimizing Capillary Electrophoresis . . . . . . . . . . . . . . . . . 67

Safety information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Thermo Fisher Scientific Genetic Analyzers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Overview of run modules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Using controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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68
68
69
69

Contents

3500 Series instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .


Run modules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Dye sets and matrix standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Creating a custom dye set . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
For more information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

69
69
70
70
70
72

3730 Series instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .


Run module and performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Dye sets and matrix standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Creating a custom dye set . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
For more information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

73
73
73
73
74

3130 Series instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .


Run modules and performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Dye sets and matrix standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Creating a custom dye set . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
For more information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

75
75
75
75
75

310 instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
Run modules and performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
Dye sets and matrix standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
Optimizing sample loading concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
Optimizing signal intensity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Optimal detection ranges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Balancing size-standard and sample-peak intensities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
If signal intensity is above the detection range . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
If signal intensity is below the detection range . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Minimizing signal intensity variation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

77
77
77
78
78
78

Optimizing electrokinetic injection parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .


Definition of resolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Optimizing injection time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Optimizing injection voltage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

78
79
79
80

Optimizing electrophoresis conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .


Optimizing run time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Optimizing run voltage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Optimizing run temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

80
81
81
82

Other factors that affect electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .


Laboratory temperature and humidity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Salt concentration, ionic strength, and conductivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Hi-Di Formamide storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Polymer handling and characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

82
82
82
82
83

Understanding spatial calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84


Understanding spectral calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Spectral calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Evaluating the calibration results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Q Value and Condition Number ranges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Troubleshooting spectral calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Understanding the matrix file (310 instruments only) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

84
85
86
87
87
87

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Contents

CHAPTER 5 Data Analysis with GeneMapper Software and Peak


Scanner Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
How the software processes data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Precise versus accurate sizing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Relative sizing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Guidelines for consistent sizing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Autoanalysis and manual analysis (GeneMapper Software only) . . . . . . . . . . . . . . . . . . . . . .

89
89
90
90
90
91

GeneMapper Software features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91


Peak Scanner Software Features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
Features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
GeneMapper Software peak detection settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Peak Amplitude Thresholds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Smoothing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Baseline Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Min. Peak Half Width . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Polynomial Degree and Peak Window Size parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Effects of varying the Polynomial Degree . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Effects of Increasing the Window Size Value . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

94
94
94
94
94
94
95
96

GeneMapper Software peak start and end settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97


How the GeneMapper Software performs sizing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
Size-standard definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
Step 1: Size matching . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
Step 2: Sizing curve and sizing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
Factors that affect sizing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
GeneMapper Software sizing methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Least Squares method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Cubic Spline Interpolation method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Local Southern method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Global Southern method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

100
100
101
102
103

Evaluating data quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .


Examining PQVs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Criteria for a good electropherogram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Examining peak definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Comparing data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

104
104
104
105
105

CHAPTER 6

Microsatellite Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . 107

Overview of microsatellite analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .


Principle of the analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Advantages of using microsatellite markers (loci) in genetic studies . . . . . . . . . . . . . . . . . .
Microsatellite motifs and distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

107
108
108
109

Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
DNA Fragment Analysis by Capillary Electrophoresis

Contents

Instrument and consumable recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111


Experiment and primer design recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
Data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
Common problems with microsatellite analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
Identifying stutter products in microsatellite analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Estimating the amount of stutter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Dinucleotide repeats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Evaluating data with stutter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Is stutter a real problem? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

113
113
114
115
117
118

For more information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118

CHAPTER 7

Single Nucleotide Polymorphism (SNP) Genotyping . . 119

Overview of SNP genotyping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119


Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
Applications (SNP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
SNaPshot Multiplex System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
Principle of the analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
Advantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
Applications (SNaPshot) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
Instrument and consumable recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122
Experiment and primer design recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
Data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
For more information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123

CHAPTER 8

Fingerprinting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125

Amplified fragment length polymorphism (AFLP) Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .


Principle of the analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Advantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Instrument and consumable recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Experiment and primer design recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
For more information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

126
126
126
127
127
127
128
129
130

Terminal restriction fragment length polymorphism (T-RFLP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . .


Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Principle of the analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

131
131
131
131

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Contents

Instrument and consumable recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .


Experiment and primer design recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
For more information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

131
132
132
132
132

Bacterial Artificial Chromosome (BAC) fingerprinting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .


Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Principle of the analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Instrument and consumable recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Experiment and primer design recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
For more information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

132
132
133
133
134
134
135
135
136

High coverage expression profiling (HiCEP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .


Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Principle of the analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
For more information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

136
136
136
136
136
136
137

Inter-simple sequence repeat (ISSR) PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .


Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Principle of the analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Advantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Experiment and primer design considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
For more information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

137
137
137
138
138
138
139
139
139
141

CHAPTER 9

Relative Fluorescence Quantitation (RFQ) . . . . . . . . . . 143

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
Principle of the analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144
Experiment and primer design recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144
Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144
Minimizing signal intensity variation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144
LOH workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
Data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Precise peak detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Determining relative quantities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Determining relative number of molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

145
145
145
146

For more information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146


Microsatellite Instability (MSI) and Replication Error (RER) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146
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Contents

CHAPTER 10

Additional Applications . . . . . . . . . . . . . . . . . . . . . . . . . . 149

DNA methylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149

CHAPTER 11

Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151

Troubleshooting workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152

10

Checking data quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .


Sizing Quality (SQ) PQV description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Checking samples with yellow and red SQ samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Examining the raw data for red SQ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Examine the sample info, raw data, and EPT trace . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

153
153
153
154
154

Running controls to isolate a problem . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .


Size standard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Installation standard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Agarose gel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
DNA template control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

156
156
156
157
157

Sample issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Sample concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Sample contamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Salt concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

158
158
158
158

Reagent and consumable issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .


Laboratory water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
PCR reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Hi-Di formamide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Polymer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Size standard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Ionic buffer strength
(not applicable to 3500 Series instruments) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

159
159
159
159
159
160
160

Instrument and ambient condition issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .


Capillary array . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Pump: large bubbles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Pump: small bubbles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Pump: polymer leaks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Autosampler misalignment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Temperature/humidity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Matrix/spectral Issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

160
160
160
161
161
161
161
161

Symptoms you may observe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .


Irregular signal intensity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Migration issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Abnormal peak morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Extra peaks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Irregular baseline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Instrumentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Sizing or size quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

162
162
162
162
162
162
163
163
163

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Contents

GeneMapper Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163


Irregular signal intensity troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164
Migration troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
Abnormal peak morphology troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
Extra peaks troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172
PCR troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176
Irregular baseline troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178
Instrumentation troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180
Sizing or Size Quality (SQ) troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Viewing the size-standard definition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Modifying the size-standard definition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

182
182
182
183

GeneMapper Software troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187


Preamplification gel troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190
Desalting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Impact of high salt concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Eliminating salt concentration as the cause . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Desalting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

190
190
190
190

Evaluating 310 Genetic Analyzer multicomponent matrix quality . . . . . . . . . . . . . . . . . . . . . . . . . .


Purpose of the multicomponent matrix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Factors affecting matrix quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
When to create a new matrix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Virtual Filter Set C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Identifying matrix problems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

191
191
191
191
191
191

Ordering Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193


Thermal cyclers and accessories . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193
Genetic analyzers and consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194
GeneScan size standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197
Matrix standards for spectral calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197
Installation standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197
Reagent kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198
Other user-supplied materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198

Documentation and Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199


Instrument documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199
GeneMapper Software documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199
Peak Scanner Software documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200
Application documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200
Obtain SDSs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201
Obtain support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201

DNA Fragment Analysis by Capillary Electrophoresis

11

Contents

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203
Glossary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211

12

DNA Fragment Analysis by Capillary Electrophoresis

About This Guide

IMPORTANT! Beforeusingtheproductsdescribedinthisguide,readandunderstand
theinformationintheSafetyappendixinthedocumentsprovidedwitheach
product.

Revision history
Revision
A

Date
August 2012

Description
New document.

Purpose
Thisguideisintendedforcustomerswhoplan,conduct,andtroubleshootfragment
analysisapplications.
Thisguideisforusebynoviceandexperienceduserswhoperformautomated
fragmentanalysiswithanyoftheseinstruments:
AppliedBiosystems3500or3500xLGeneticAnalyzers(3500Seriesinstruments)
AppliedBiosystems3730or3730xlDNAAnalyzers(3730Seriesinstruments)
AppliedBiosystems3130or3130xlGeneticAnalyzers(3130Seriesinstruments)
310GeneticAnalyzers(310instruments)

Prerequisites
Thisguideassumesthat:
ThermoFisherScientificgeneticanalyzersandotherinstrumentsforwhich
ThermoFisherScientificprovidesinstallationservicehavebeeninstalledbya
ThermoFisherScientifictechnicalrepresentative.
ThermoFisherScientificreagentsareused.

DNA Fragment Analysis by Capillary Electrophoresis

13

About This Guide


Structure of this guide

Structure of this guide


Chapter

Subject

Introduction
1

Introduction to Fragment Analysis

Experimental Design

Core processes in fragment analysis


3

Optimizing PCR

Optimizing Capillary Electrophoresis

Data Analysis with GeneMapper Software and Peak


Scanner Software

Types of fragment analysis


6

Microsatellite Analysis

Single Nucleotide Polymorphism (SNP) Genotyping

Fingerprinting

Relative Fluorescence Quantitation (RFQ)

10

Additional Applications

Troubleshooting
11

Troubleshooting

Reference information
Ordering Information
Documentation and Support
References
Glossary

14

DNA Fragment Analysis by Capillary Electrophoresis

Introduction to Fragment Analysis

Fragmentanalysisversussequencingwhatisthedifference? .............. 15

WhatcanIdowithfragmentanalysis? ................................... 16

Whatiscapillaryelectrophoresis?....................................... 18

Fragmentanalysisworkflow ........................................... 19

Fragment analysis versus sequencingwhat is the difference?


Fragment analysis

FragmentanalysisusingThermoFisherScientificproductsinvolves:
Labelingfragmentswithfluorescentdyes.Multipledifferentcoloredfluorescent
dyescanbedetectedinonesample.Oneofthedyecolorsisusedforalabeledsize
standardpresentineachsample.Thesizestandardisusedtoextrapolatethe
basepairsizesofthesampleproductpeaks.
Amplifyingthelabeledfragmentsusingpolymerasechainreaction(PCR)ona
thermalcycler.
Separatingthefragmentsbysizeusingcapillaryelectrophoresis.
Analyzingthedatausingsoftwaretodetermine:
Size:Theanalysissoftwareusesthesizestandardineachsampletocreatea
standardcurveforeachsample.Itthendeterminestherelativesizeofeach
dyelabeledfragmentinthesamplebycomparingfragmentswiththe
standardcurveforthatspecificsample.
Genotype:Theanalysissoftwareassignsallelecallsbasedonuserdefined
makers(loci).
Figure1 Fragment analysis fluorescently labeled fragments are separated and sized

Intensity (RFU)

Size (bp)

DNA Fragment Analysis by Capillary Electrophoresis

15

Chapter1 Introduction to Fragment Analysis


What can I do with fragment analysis?

Sequencing

SequencingisthedeterminationofthebasepairsequenceofaDNAfragmentbythe
formationofextensionproductsofvariouslengthsamplifiedthroughPCR.Formore
information,refertotheDNASequencingbyCapillaryElectrophoresis|ChemistryGuide
(Pub.no.4305080).
Figure2 Sequencing fluorescently labeled nucleotides are separated and base-called

Intensity (RFU)

Base assignment

What can I do with fragment analysis?


Types of
applications

Microsatellite(STR)analysis(seeChapter6,MicrosatelliteAnalysis)
Microsatellitemarkers(loci),alsoknownasshorttandemrepeats(STRs),are
polymorphicDNAlociconsistingofarepeatednucleotidesequence.Inatypical
microsatelliteanalysis,microsatellitelociareamplifiedbyPCRusing
fluorescentlylabeledforwardprimersandunlabeledreverseprimers.ThePCR
ampliconsareseparatedbysizeusingelectrophoresis.Applicationsinclude:
Linkagemapping
Animalbreeding
Human,animal,andplanttyping
Pathogensubtyping
Geneticdiversity
Microsatelliteinstability
LossofHeterozygosity(LOH)
Intersimplesequencerepeat(ISSR)
MultilocusVariantAnalysis(MLVA)
SNPGenotyping(seeChapter7,SingleNucleotidePolymorphism(SNP)
Genotyping)
ASingleNucleotidePolymorphism(SNP)markerconsistsofasinglebasepair
thatvariesintheknownDNAsequence,therebycreatinguptofourallelesor
variationsofthemarker.Applicationsinclude:
SNaPshotMultiplexKit

16

DNA Fragment Analysis by Capillary Electrophoresis

Chapter1 Introduction to Fragment Analysis


What can I do with fragment analysis?

Fingerprinting(seeChapter8,Fingerprinting)
SeveralAFLPbasedtechnologiesuserestrictionenzymelengthpolymorphism
andpolymerasechainreaction(PCR)togenerateafingerprintforagivensample,
allowingdifferentiationbetweensamplesofgenomicDNAbasedonthe
fingerprint.Applicationsinclude:
Microbialgenometyping
Animalorplantgenometyping
Creationofgeneticmapsofnewspecies
Geneticdiversityandmolecularphylogenystudies
Establishmentoflinkagegroupsamongcrosses
RelativeFluorescence(seeChapter9,RelativeFluorescenceQuantitation
(RFQ))
Relativefluorescenceapplicationscomparepeakheightorareabetweentwo
samples.Commontechniquesinclude:
QualitativeFluorescence(QF)PCR
QuantitativeMultiplexPCRofShortFluorescentFragments(QMPSF)
MultiplexLigationdependentProbeAmplification(MLPA)
Applicationsinclude:
LOHintumorsamples
CopyNumberVariation(CNV)
Aneuploidydetection

Applications
described in this
guide

Theapplicationsinthisguideareidentifiedasoneofthefollowing:

Category

Description

Thermo Fisher Scientific-supported

Thermo Fisher Scientific has tested and validated this protocol on the instrument
system specified. The technical support and field application specialists have
been trained to support this protocol.

Thermo Fisher Scientificdemonstrated

Thermo Fisher Scientific has tested this protocol but has not validated for the
instrument system specified. Certain components of the protocol workflow such
as reagent kits and other protocols for preparation of reagents may not be
available through Thermo Fisher Scientific. Supporting documentation such as
application notes may be available from Thermo Fisher Scientific and/or third
parties. Limited support is available from Thermo Fisher Scientific.

Customer-demonstrated

Thermo Fisher Scientific has not tested this protocol. However, at least one
customer or third party has reported successfully performing this protocol on the
instrument system specified. Thermo Fisher Scientific cannot guarantee
instrument and reagent performance specifications with the use of customerdemonstrated protocols. However, supporting documentation from Thermo
Fisher Scientific and/or third parties may be available and Thermo Fisher
Scientific may provide basic guidelines in connection with this protocol.

DNA Fragment Analysis by Capillary Electrophoresis

17

Chapter1 Introduction to Fragment Analysis


What is capillary electrophoresis?

What is capillary electrophoresis?


Capillaryelectrophoresis(CE)isaprocessusedtoseparateionicfragmentsbysize.In
ThermoFisherScientificCEinstrumentation,anelectrokineticinjectionisusedto
injectDNAfragmentsfromsolutionandintoeachcapillary.
Duringcapillaryelectrophoresis,theextensionproductsofthePCRreaction(andany
othernegativelychargedmoleculessuchassaltorunincorporatedprimersand
nucleotides)enterthecapillaryasaresultofelectrokineticinjection.Ahighvoltage
chargeappliedtothesampleforcesthenegativelychargedfragmentsintothe
capillaries.Theextensionproductsareseparatedbysizebasedontheirtotalcharge.
Theelectrophoreticmobilityofthesamplecanbeaffectedbytherunconditions:the
buffertype,concentration,andpH;theruntemperature;theamountofvoltage
applied;andthetypeofpolymerused.
Shortlybeforereachingthepositiveelectrode,thefluorescentlylabeledDNA
fragments,separatedbysize,moveacrossthepathofalaserbeam.Thelaserbeam
causesthedyesattachedtothefragmentstofluoresce.Thedyesignalsareseparated
byadiffractionsystem,andaCCDcameradetectsthefluorescence.
Capillary array

CCD
camera
Diffraction
system

Laser

Becauseeachdyeemitslightatadifferentwavelengthwhenexcitedbythelaser,all
colors,andthereforeloci,canbedetectedanddistinguishedinonecapillaryinjection.
Thefluorescencesignalisconvertedintodigitaldata,thenthedataisstoredinafile
formatcompatiblewithananalysissoftwareapplication.

18

DNA Fragment Analysis by Capillary Electrophoresis

Chapter1 Introduction to Fragment Analysis


Fragment analysis workflow

Fragment analysis workflow


Phase

Technology

Thermo Fisher Scientific products used

1.

Isolate DNA

Depends on sample source


and application

DNA isolation methods depend on your starting DNA source.


Refer to guidelines for your application for information on
isolating DNA.

2.

Purify DNA

Depends on sample source


and application

Go to www.lifetechnologies.com for advice on the


appropriate product to use.

3.

Quantify DNA

Dye-labeling and
fluorometric detection

Qubit Fluorometer and Quantitation Kit, go to


www.lifetechnologies.com/qubit.

4.

PCR
amplification

Dye-labeling and
amplification of fragments
using a thermal cycler

Veriti Thermal Cycler:


96-well
384-well
GeneAmp PCR System 9700:
Dual 96-well
Dual 384-well
Auto-Lid Dual 384-well
2720 Thermal Cycler

5.

Capillary
electrophoresis

Separation of fragments
based on size using a genetic
analyzer

3500/3500xL Genetic Analyzer (3500 Series instrument)


3730/3730xl Genetic Analyzer (3730 Series instrument)
3130/3130xl Genetic Analyzer (3130 Series instrument)
310 Genetic Analyzer (310 instrument)

6.

Data analysis

Sizing and optional


genotyping

GeneMapper Software

Sizing

Peak Scanner Software (available free-of-charge on


www.lifetechnologies.com)
Use this software with data generated on 3730 Series,
3130 Series, and 310 instruments. It is not compatible
with data generated on the 3500 Series instrument, which
performs fragment sizing during data collection.

DNA Fragment Analysis by Capillary Electrophoresis

19

20

Chapter1 Introduction to Fragment Analysis


Fragment analysis workflow

DNA Fragment Analysis by Capillary Electrophoresis

Experimental Design

Experimentaldesignconsiderations..................................... 21

DNApolymeraseenzymes ............................................. 22

Fluorescentlabelingmethods ........................................... 25

Singleplexingversusmultiplexing....................................... 26

Primerdesignguidelines............................................... 29

Dyes ................................................................ 36

Dyesets ............................................................. 41

Sizestandards ........................................................ 42

Orderingcustomprimers .............................................. 52

TestingtheprimersandoptimizingconditionswithtestDNApanel ......... 52

Experimental design considerations


Considerthefollowingquestionswhendesigningyourexperiment:
Whatsequencesandmarkers(loci)areyouinvestigating?(notapplicablefor
AFLPstudies)
Whichenzymeisappropriateforyourexperiment?(seeDNApolymerase
enzymesonpage 22)
Whatistheexpectedalleledistribution?(determinefrompublishedliteratureor
fromyourowndesignandempiricaltesting)
Whatlabelingmethodwillyouuse?(seeFluorescentlabelingmethodson
page 25)
Dofragmentsizesoverlap?(seeCompensatingforoverlappingfragmentsizes
onpage 28)
Willyouevaluateonetargetperreaction(singleplex)ormultipletargetsper
reaction(multiplex)?(seeSingleplexingversusmultiplexingonpage 26)
Whatfactorsaffectthedesignofyourprimers?(seePrimerdesignguidelines
onpage 29)
Whichdyesetsarecompatiblewithyourgeneticanalyzerandareappropriatefor
thenumberofmarkersofinterest?(seeDyesetsonpage 41andSingleplexing
versusmultiplexingonpage 26)
Whichsizestandardisappropriateforthefragmentsizerangeanddyelabelsof
yoursamples?(seeSizestandardsonpage 42)

DNA Fragment Analysis by Capillary Electrophoresis

21

Chapter2 Experimental Design


DNA polymerase enzymes

DNA polymerase enzymes


Overview

Formostapplications,AmpliTaqGoldDNAPolymeraseistheenzymeofchoice.
However,ThermoFisherScientificsuppliesanumberofPCRenzymesthathavebeen
optimizedforspecificneedsaslistedbelow.Gotowww.lifetechnologies.comfor
otheravailableenzymes.
Note: AmpFlSTR,AFLP,andSNaPShotkitsincludetheappropriateDNA
polymerasefortheapplication.
Table1 PCR enzymes supplied by Thermo Fisher Scientific

DNA polymerases

Description

AccuPrime

Taq
DNA Polymerase
System

Provides reagents for amplification of nucleic acid templates with antibody-mediated hot-start for
improved PCR specificity over other hot-start DNA polymerases. Platinum anti-Taq DNA
polymerase antibodies inhibit polymerase activity, providing an automatic hot-start, while a
thermostable accessory protein enhances specific primer-template hybridization during every
cycle of PCR. This combination improves the fidelity of Taq DNA Polymerase by two-fold and is
ideal for high-throughput screening and multiplex PCR. AccuPrime Taq DNA Polymerase
broadens primer annealing temperatures, giving you optimal performance between 55C and
65C. Applications: Multiplex PCR, TOPO TA Cloning, allele-specific amplifications.

AccuPrime Taq
DNA Polymerase
High Fidelity

Amplifies nucleic acid templates using antibody-mediated hot-start, a blend of Taq DNA
Polymerase and proofreading enzyme, and AccuPrime accessory proteins for improved PCR
fidelity, yield, and specificity over other hot-start DNA polymerases. This enzyme provides:
The highest specificity and yield for the most robust PCR amplification
9-fold higher fidelity than Taq DNA polymerase alone
Minimal optimization steps, even with non-optimized primer sets
Efficient amplification of targets over a broad size range up to 20 kb
High fidelity is achieved by a combination of Platinum anti-Taq DNA polymerase antibodies that
inhibit polymerase activity, providing an automatic hot-start, and the proofreading (3-5
exonuclease activity) enzyme Pyrococcus species GB-D. The thermostable AccuPrime accessory
proteins enhance specific primer-template hybridization during every cycle of PCR, preventing
mispriming and enhancing PCR specificity and yield.

AmpliTaq DNA
Polymerase

For general use in PCR.

AmpliTaq DNA
Polymerase, LD

Low concentrations of E. coli DNA contamination, thus is better suited for amplifying DNA of
bacterial origin.

AmpliTaq DNA Polymerase is a recombinant form of Taq DNA polymerase obtained by


expressing a modified Taq DNA polymerase gene in an E. coli host. Similar to native Taq DNA
polymerase, the enzyme lacks endonuclease and 3'-5' exonuclease (proofreading) activities, but
has a 5'-3' exonuclease activity.

AmpliTaq DNA Polymerase, LD (Low DNA), is the same enzyme as AmpliTaq DNA Polymerase;
however, the LD formulation has undergone a further purification process. The purification step
insures that false-positive PCR products will be effectively minimized when amplifying bacterial
sequences. AmpliTaq DNA Polymerase, LD is especially useful for low-copy number
amplifications.

22

DNA Fragment Analysis by Capillary Electrophoresis

Chapter2 Experimental Design


DNA polymerase enzymes

DNA polymerases
AmpliTaq Gold
DNA Polymerase

Description
Use in most applications because it yields PCR fragments of high specificity.
AmpliTaq Gold DNA Polymerase is a chemically modified form of AmpliTaq DNA Polymerase.
It provides the benefits of hot-start PCR (that is, higher specific product yield, increased sensitivity,
and success with multiplex PCR) without the extra steps and modifications of experimental
conditions that make hot-start impractical for high-throughput applications. AmpliTaq Gold DNA
Polymerase is delivered in an inactive state. A pre-PCR heating step of 10 to 12 minutes at 95C,
which can be programmed into the thermal cycling profile, activates the enzyme. For low-template
copy number amplifications, step-wise activation of AmpliTaq Gold DNA Polymerase, or
time-release PCR, can be useful.

GeneAmp Gold
Fast PCR Master Mix

Allows PCR to be finished in ~40 minutes.

Platinum Multiplex
PCR Master Mix

Designed specifically for endpoint multiplex PCR. It supports easy multiplexing with minimal
optimization. Amplifies up to 20 amplicons in a single reaction. Amplifies products from
50 bp to 2.5 kb.
The performance of the Platinum Multiplex PCR Master Mix over a wide range of amplicon sizes
permits the amplification of templates from 50 bp to 2.5 kb, greatly enhancing workflow flexibility.
Coupled with its 20-plex capability and absence of primer dimers, it not only provides a
high-throughput solution but also boasts high specificity through fewer non-specific primer
binding events, and hence less reaction and primer waste.

Platinum Pfx DNA


Polymerase

Ideal for amplification of DNA fragments for high-fidelity PCR applications. High fidelity is provided
by a proprietary enzyme preparation containing recombinant DNA Polymerase from Thermococcus
species KOD with proofreading (3t exonuclease) activity. Platinum antibody technology provides
a simple, automatic hot-start method that improves PCR specificity. PCRx Enhancer Solution is
included for higher primer specificity, broader magnesium concentration, broader annealing
temperature, and improved thermostability of Platinum Pfx DNA Polymerase. The PCRx
Enhancer Solution also helps optimize PCR of problematic and/or GC-rich templates. Platinum
Pfx provides:
26 times higher fidelity than Taq DNA polymerase
Amplification of fragments up to 12 kb
Room temperature reaction assembly
Applications: Amplification of DNA from complex genomic, viral, and plasmid templates; and
RT-PCR.
Unit Definition: One unit incorporates 10 nmoles of deoxyribonucleotide into acid-precipitable
material in 30 minutes at 74C.

SuperScript III
Reverse
Transcriptase (RT)

Proprietary mutant of SuperScript II RT that is active at 50C and has a half-life of 220 minutes,
providing increased specificity with Gene-Specific Primers (GSPs) and the highest cDNA yield of
all RTs. It is ideal for RT-PCR of a specific gene or generating cDNA from total or poly (A)+ RNA
sample. Like SuperScript II, it synthesizes a complementary DNA strand from single-stranded
RNA, DNA, or an RNA:DNA hybrid. SuperScript III RT is genetically engineered by the
introduction of point mutations that increase half-life, reduce RNase activity, and increase thermal
stability. Applications: array labeling, cDNA libraries, RT-PCR, primer extension, and 3 and 5
RACE. Purified from E. coli.

DNA Fragment Analysis by Capillary Electrophoresis

23

Chapter2 Experimental Design


DNA polymerase enzymes

Derivatives of Tth
DNA polymerase

ThermoFisherScientificsuppliestwomodifiedformsofThermusthermophilus(Tth)
DNApolymerase:
rTthDNAPolymeraseisobtainedbyexpressionofamodifiedformoftheTth
geneinanE.colihost.
rTthDNAPolymerase,XL(ExtraLong),providesthesamefeaturesasrTthDNA
Polymerasefortargetsequencesfrom5to40kb.Aninherent35exonuclease
activityallowsforthecorrectionofnucleotidemisincorporationsthatmight
otherwiseprematurelyterminatesynthesis.

Enzyme
characteristics

Table2 Enzyme characteristics


Characteristics
High specificity

Recommended enzyme
AccuPrime Taq DNA Polymerase

High sensitivity

AmpliTaq Gold DNA Polymerase

High fidelity

Platinum Pfx DNA Polymerase

High temperatures

AmpliTaq DNA Polymerase

Multiplex PCR

Platinum Multiplex PCR Master Mix

Amplification of low-copy number template

AmpliTaq Gold DNA Polymerase


AmpliTaq DNA Polymerase
AmpliTaq DNA Polymerase LD (for
bacterial sequences)

High specificity at high ionic strength

AmpliTaq Gold DNA Polymerase


AmpliTaq DNA Polymerase

Amplification of extra-long fragments


(>5 kb)

rTth DNA Polymerase, XL

Pre-PCR conversion to cDNA

SuperScript III Reverse Transcriptase

Extra cycles

AmpliTaq DNA Polymerase

High magnesium ion concentration

AmpliTaq Gold DNA Polymerase


AmpliTaq DNA Polymerase

24

DNA Fragment Analysis by Capillary Electrophoresis

Chapter2 Experimental Design


Fluorescent labeling methods

Fluorescent labeling methods


IMPORTANT! Withanylabelingtechnique,useonlyThermoFisherScientificdyes.
ThermoFisherScientificprovidesspectralcalibrationmatrixstandardsthathavebeen
optimizedforourdyesets.
Otherdyes(ormixedisomersofdyes)havevariableemissionspectraandrequirea
spectralcalibrationgeneratedforthespecificdyestocorrectforthespectraloverlap
betweenthedyes.Youareresponsibleforobtainingtheappropriatespectral
calibrationreagentsandforoptimizingcustomdyesets.
Table3 Fluorescent labeling methods
5'-end labeled primer
incorporated during the PCR primer-annealing step

Fluorescent dye-labeled dUTPs or dCTPs ([F]dNTPs


incorporated during the PCR primer-extension step

Most commonly used in microsatellite analysis

Most commonly used in SNP analysis

Higher precision: Different fluorophores have different


mobilities. DNA fragments with the same 5'-end primer
and fluorophore have comparable electrophoretic
mobility, and yield sharper fragment peaks because
5-end primer labeling yields 1:1 incorporation (that is,
one fluorophore-to-one DNA fragment).

Lower precision: Fragments labeled with [F]dNTPs tend


to produce broader peaks that often appear to be split
because variable numbers of [F]dNTPs are incorporated
during PCR in variable positions on both strands.

More consistent quantitation: Every peak in an


electropherogram is made up of multiple DNA
fragments of equal size in base pairs. When using
5'-end primer labeling, every DNA fragment contributes
a single fluorophore to the total signal of a peak, and
thus the peak area is proportional to the number of DNA
molecules.
Distinct strands: By attaching different fluorophores to
the forward and reverse primers, it is possible to
distinguish between the peaks corresponding to each
strand, and between residual double-stranded products.
Lower sensitivity: Because of 1:1 incorporation (that is,
one fluorophore-to-one DNA fragment), it yields a lower
signal than [F]dNTP-labeled fragments.

Less consistent quantitation: A variable number of


fluorophores are attached to each DNA fragment in a
population. The average number of attached
fluorophores depends upon the fragment base
composition and length and upon the ratio of [F]dNTPs
to dNTPs added to the reaction mixture. Therefore, it is
not advisable to compare peak areas between
fragments labeled with [F]dNTPs for relative
quantitation studies.
Higher sensitivity: Because most fragments contain
multiple fluorophores, a given number of [F]dNTPlabeled fragments will produce a higher signal when
compared to the same number of 5'-end labeled
fragments. The increased signal strength allows you to
use smaller reaction volumes and fewer amplification
cycles during PCR.
Low cost: You can add [F]dNTPs to any PCR. You do not
need to order or synthesize fluorescently labeled
primers before each PCR and you can use [F]dNTPs
with your existing primer sets.

5-end labeled primer

Labeled nucleotides

Post-PCR end-labeling with [F]dNTPs using Klenow is an alternative to labeling during PCR (Iwahana et al., 1995; Inazuka et al., 1996). You can
also label with [F]dNTPs using traditional techniques such as random priming or nick translation.

DNA Fragment Analysis by Capillary Electrophoresis

25

Chapter2 Experimental Design


Singleplexing versus multiplexing

Thefollowingfigurecomparestheresultsobtainedusing5endlabeledprimersand
[F]dNTPs.The5endlabeledprimersgivebetterresolution,but[F]dNTPsresultin
higherpeaks.Notealsotheunincorporatedfluorescentlylabelednucleotidesinthe
[F]dNTPlabeledsample.
Figure3 Comparison of 5'-end labeled primers (top panel) and [F]dNTP-labeled primers
(bottom panel)

Singleplexing versus multiplexing


Singleplexing

SingleplexingisaPCRtechniqueinwhichasingletargetisamplifiedinareaction
tube.Thistechniqueusesonlyoneprimerpairineachreactionanddoesnotrequireas
muchoptimizationasmultiplexing.However,singleplexingincreasesthecostand
timeperanalysis.

Multiplexing

MultiplexingisaPCRtechniqueinwhichmultipleDNAtargetsareamplifiedinthe
samereactiontube.Multiplexingusesmultipleprimerpairsineachreaction,and
requiresoptimizationtoensureprimerpairsarecompatible.
ThermoFisherScientificfluorescentmulticolordyetechnologyallowsmultiplexing.
Allelesforoverlappinglociaredistinguishedbylabelinglocusspecificprimerswith
differentcoloreddyes.Multicomponentanalysisseparatesthedifferentfluorescent
dyecolorsintodistinctspectralcomponents.
Benefits

Potential limitations

Simplifies PCR setup

Primer-oligomer formation

Increases throughput

Loss of specificity

Decreases cost per amplification

Decreased yield of specific products


Can require significant optimization

26

DNA Fragment Analysis by Capillary Electrophoresis

Chapter2 Experimental Design


Singleplexing versus multiplexing

Multiplexing
(pooling) strategies

Strategies
Multiplexingstrategiesinclude:
PoolingsamplesafterPCR
Note: Itisgenerallyeasiertopooltheproductsfromindividuallyamplified,
fluorescentlylabeledprimerpairsthantooptimizeamultiplexPCRcontaining
multiplefluorescentlylabeledprimerpairs.Becauseprimerefficienciesvary,it
maybenecessarytoadddifferentamountsofeachindividuallyamplifiedPCR
producttoapooltoachievesimilarpeakheights.Fluorescenceintensityfrom
eachindividualdyemayalsovary.
AmplifyingmultipleproductsinasinglePCRreaction.Optionsforpoolingina
PCRreactionareillustratedinthefollowingfigure(theorangepeaksarethe
sizestandardpeaks).

1 singleplex
reaction

1 multiplex
reaction

>1 singleplex
reactions

If you pool samples after PCR:


This strategy is simpler and more flexible, but pooling
products from multiple singleplex PCR reactions often
increase the salt concentration in the loaded samples,
which can cause unwanted downstream effects (see
Desalting on page 190).
If PCR product sizes overlap, use different color dyes so
they separate during electrophoresis (see Dyes on
page 36).
Use a combination of dyes that can be detected using
one spectral matrix (one spectral calibration).
Optimize sample concentration to optimize signal
intensity for each dye (see Optimizing signal intensity
on page 77).

DNA Fragment Analysis by Capillary Electrophoresis

If you amplify multiple PCR products:


Optimize primers:
Use different dyes to label multiplex primers of
similar lengths.
Primers cannot contain large regions of
complementarity.
Primers should have similar melting temperatures
(Tm).
Before performing the PCR, perform a preliminary
check for primer compatibility and test the pairs for
successful co-amplification.
Optimize conditions for primers in singleplex reactions
before using them in multiplex reactions to ensure the
primers are suitable for your experiment.

27

Chapter2 Experimental Design


Singleplexing versus multiplexing

Adjusting pooling ratios


Toensuresignalbalancebetweendyesinamultiplexedsample,adjustpoolingratios
asneeded.Thefigurebelowshowstheeffectofdifferentpoolingratiosonsignal
balance.Inthisexample,apoolingratioof3:1:1yieldsbalancedsignalforthethree
dyes.
1:1:3

3:1:1

1:3:1

3:1:3

1:3:3

3:3:1

Multiplex design
software

SoftwareapplicationsareavailabletoassistwiththedesignofmultiplexPCR
(HolleleyandGeerts,2009).

Multiplexing
guidelines

Compensating for overlapping fragment sizes


Ifthesizesofdifferentfragmentsoverlap,youcandothefollowingtodifferentiate
betweenthem:
Labeloverlappingproductswithdifferentdyes.
Leavethefollowingnumberofbasepairsbetweentheknownsizeranges:
Microsatelliteapplications:15to20basepairs
SNaPshotapplications:8to10basepairs
UsedifferentprimersitestoalterthePCRproductfragmentlengths.
Loadoverlappingproductsindifferentwellsorseparatecapillaryinjections
(runs).

Enzyme choice
ThehighspecificityofAmpliTaqGoldDNAPolymerasetypicallypermits
amplifyingwithelevatedMg2+concentrationsforincreasedyield.

Primer quality
Becausereagents(suchasdNTPs)areoftenlimitingduringmultiplexPCR,usinghigh
qualityprimersisparticularlyimportant.Forexample,thedecreasedspecificity(and
thustheincreasedreagentconsumption)ofonepairofdegradedPCRprimerscan
affecttheentiremultiplexreaction.Althoughyoucancompensateforadegradedpair
ofprimerstosomeextentbyincreasingtheconcentrationoftheotherprimerpairs,the
increasedcostperreactionandthedecreasedreproducibilityovertimedonotjustify
thisshorttermsolution.
Whenbuyingormakingprimers,makesurethattheyarelengthpurifiedandthat
theyarefreeofcontaminants.

28

DNA Fragment Analysis by Capillary Electrophoresis

Chapter2 Experimental Design


Primer design guidelines

Primer-pair concentrations
Typically,startoutwithequalconcentrationsforallprimerpairs.
Itisoftennecessarytoadjusttheconcentrationofprimerpairsinthemultiplex
reactionuntilthepeakheightsarerelativelyeven.Increasetheprimerpair
concentrationforfragmentsshowingweakamplification.Decreasetheprimerpair
concentrationforfragmentsshowingsignificantlygreaterthanaverageamplification.

Primer-pair compatibility
WitheithersingleormultiplexPCR,evaluateprimersforcompatibility.Avoid
excessiveregionsofcomplementarityamongtheprimers.Also,selectordesign
primerswithsimilarmeltingtemperatures(Tm).
Afteridentifyingcompatibleprimerpairs,testandevaluatepairsinsingleplex
reactionsbeforeattemptinganymultiplexreactions.Youwilloftenneedtooptimize
reactionconditionsand,occasionally,youwillneedtoredesigntheprimers.

Troubleshooting multiplex PCR


Consideramplifyingseparatelyanyprimerpairthatfailstoamplifyafterits
concentrationisincreased.
Toeliminateinterferingbackgroundpeaks,try:
Swappingprimerpairsbetweendifferentmultiplexreactions
Removingprimerpairsfromthemultiplexreaction

Primer design guidelines


Primer design
criteria

Optimumlength:17to25nucleotides
OptimumTm:55to65C
UsingprimerswithsimilarTmvaluesmakesitpossibletofindthermalcycling
parametersthatareoptimalforbothprimersinaprimerpair.TheTmofareaction
isinfluencedbybasecomposition,concentrationsofMg2+andK+ionsinthe
mixture,andcosolvents.
BasedontheTm,calculatetheannealingtemperature:
Ta(C)=Tm5
The2+4Rule:Tm=[(A+T)2+(G+C)4]
Avoid:
Primerdimers
Hairpins
Secondarystructures
Secondarybindingsites

Primer design
software

ThermoFisherScientificoffersOligoPerfectDesigneravailableat
www.lifetechnologies.com.

DNA Fragment Analysis by Capillary Electrophoresis

29

Chapter2 Experimental Design


Primer design guidelines

Factors affecting
Tm and primer
annealing

Primerannealingisinfluencedby:
Primer/templatebasecomposition
Primer/templatebaseorder
Primerortemplatesecondarystructure

Effects of base composition


GCbondscontributemoretothestability(increasedmeltingtemperature)ofprimer/
templatebindingthandoATbonds.
Toensurestableannealingofprimerandtemplatewhileavoidingproblemswiththe
internalsecondarystructureofprimersorlongstretchesofanyonebase,select
primerswitha40%to60%G+Ccontent.
Note: DesigningprimersbasedonTmandprimerlengthtoavoidprimerdimersand
gappedduplexstructuresismoreimportantthandesigningprimersbasedonactual
percentG+Ccontent.

Effects of base order


Twoprimer/templatecomplexeswithidenticalcontentmayhavedifferentTmvalues
becausebaseorderinfluencestheoverallannealingstability.Youcandeterminethe
exacteffectofbaseorderoncomplexstabilityusingthebasepairingenergieslistedin
Table4(adaptedfromSalser,1978).Largernegativevaluesrepresentmorestable
interactionsbetweenthetemplateandprimer.
Table4 Base-pairing energies (kcal/dinucleotide pair)
3' Nucleotide

5' Nucleotide
A

1.2

2.1

2.1

1.8

2.1

4.8

3.0

2.1

2.1

4.3

4.8

2.1

1.8

2.1

2.1

1.2

Example:Considerthetwosequences:3GAC5and3CGA5.Thesequence
3GAC5containedwithinaprimerwouldcontribute 4.2kcaltothebindingenergy
( 2.1kcal[3GA5]+ 2.1kcal[3AC5]= 4.2kcal).However,iftheGandCarenext
toeachother,asin3CGA5,thecontributionincreasesto 6.4kcal
( 4.3kcal[3CG5]+ 2.1kcal[3GA5]= 6.4kcal).
Note: AlthoughaGCdinucleotideatthe3endoftheprimercanstabilizethe
templateprimerbindingcomplexwhenusingthermostableenzymessuchas
AmpliTaqDNAPolymerase,a3GCcanalsoleadtofalseprimingifyoudonot
optimizePCRconditions(TopalandFresco,1976).

Effects of primer secondary structure


StringsofGsandCscanforminternal,nonWatsonCrickbasepairs(Sarocchietal.,
1970)thatdisruptstableprimerannealing.Althoughthisanomalousbehavioris
difficulttopredict,agoodgeneralruleistoavoidrunsofmorethanthreeconsecutive
Gsinprimers.

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DNA Fragment Analysis by Capillary Electrophoresis

Chapter2 Experimental Design


Primer design guidelines

However,ashortrunofGsatornearthe5endofaprimerwillnotdisruptthe
stabilityofprimertemplatecomplexesbecause5positioningdoesnotleadto
involvementindisruptivesecondarystructures(forexample,primerdimerorduplex
loops).
Similarly,selfcomplementarysequenceswithintheprimercanleadtotheformation
ofhairpinstructuresthatdisruptstableprimerbindingtotemplate.Astablehairpin
canformwithjustfourG+Cbasepairsinthestemandthreebasesintheloop
(Summeretal.,1985)(Figure4).
Figure4 Secondary structures in primers

Forward and
reverse primers

Hairpin loop
sequence

Hairpin loop
sequence

Multiple binding sites

Effects of template
secondary
structure

Primersdonotbindeffectivelytotargetsequenceswithknownsecondarystructures.
Forexample,hairpinstructuresareoftenfoundinregionsofhighG+Ccontentorin
RNAsequences.Ifyoumustdesignprimerstoaspecifictargetregionwiththe
potentialforhairpinformation,youmaytryadditionofDMSOtoyourreactionor
othercommerciallyavailablekitsfordifficulttemplateamplification.

Selective
amplification

Amplificationofthedesiredtargetsequencerequiresminimizingprimerbindingto
secondarysitesintheDNAandtootherprimers.
Note: ThisappliestotemplategenomicDNA.Theprobabilityofbindingtosecondary
sitesislowerforlowcomplexitytemplates,suchasplasmidDNA.
Ideally,thebindingoftheprimertothedesiredtemplateregion:
Isstrongestatthe5end.
Generallyrequiresahigher,morenegativevalue(tomaximizebasepairing
energy)than
9.8kcal/moleatthe3end(seeTable4onpage30).
Asageneralrule,bindingatthe3endshouldbeweakerthan 9.8kcal/mole.

Preferential
amplification

Whenallelesdifferinsizebytenormorebasepairsyoumayobservepreferential
amplificationofshorterPCRproductsoverlongerones(Walshetal.,1992).Thiswill
alsooccurwhenamplifyinglowcopynumberDNAorDNAisolatedfrom
paraffinembeddedtissues.Figure5onpage32isanexampleofpreferential
amplificationoftheD5S346marker.Inboththenormal(toppanel)andtumor(bottom
panel)samples,thepeakheightofthelarger124basepair(bp)fragmentismuch
lowerthanthatofthesmaller110bpfragment.

DNA Fragment Analysis by Capillary Electrophoresis

31

Chapter2 Experimental Design


Primer design guidelines

IMPORTANT! Preferentialamplificationcandecreasetheaccuracyofrelative
quantitationmeasurements.
Figure5 Example of preferential amplification of the D5S346 marker. In both the normal (top
panel) and tumor (bottom panel) samples, the peak height of the larger 124-bp fragment is
much lower than that of the smaller 110-bp fragment.

Non-specific
amplification

Polymerasesrequireonlythebindingofthenucleotidesatthe3endtobegin
elongation.Ifthe3nucleotidesbindstronglytorandomregionsofthegenome
(perhapsbecauseofa3G+C),anytemplatesequencescomplementarytothe3endare
amplified.Inthiscase,specificityislostbecausetheentireprimerdoesnotspecifically
targetthegenomicregionofinterest.
Selfcomplementarysequenceswithintheprimercanleadtotheformationofhairpin
structuresthatdecreasebindingspecificity(aswellasdisruptbindingstability).
Nucleotidesinthehairpinstructurearenotavailableforbindingofthetarget
sequence.Theavailablenucleotidescanbethoughtofasformingasmaller,and
thereforelessspecific,primer.
Conversely,ifbindingisstrongestatthe5end,thetypicalbindingeventonthe
templateDNAbeginsatthe5end.Polymerases,however,cannotbeginelongation
untilthe3endbinds.Therefore,theentireprimerisusedtodistinguishamongtarget
sequences.
Also,whenperformingacomputerassistedsearchtoevaluatebindingtosecondary
sitesinthetargetDNA,considerthepotentialforgappedduplexformation.A
gappedduplexcanformwhentheprimerandtargetarecompletelycomplementary
exceptforasinglebase(Miller,Kirchoffetal.,1987;Miller,Wlodaweretal.,1987).
Note: Bindingtosecondarysitescanalsoinvolvetheformationofstable
nonWatsonCrickbasepairs(TopalandFresco,1976).Stablebasepairingismost
likelytooccurbetweenGandT,butACandGApairscanalsobestable(Hunter,
1986).Allsoftwareprogramshavedifficultymodelingthesesortsofinteractions.

Minimizing binding
to other primers

32

Complementarysequencesbetweentwoprimers,especiallyatthe3ends,canleadto
theformationofproductartifactsarisingfromamplifiedprimerdimersand
primeroligomers.Avoidprimerswithintercomplementaryregionsbetween
membersofaprimerpairorpairs.

DNA Fragment Analysis by Capillary Electrophoresis

Chapter2 Experimental Design


Primer design guidelines

Post-amplification
manipulations

Addingextensionsthatarenotcomplementarytothetemplateatthe5endofthe
primercanfacilitateavarietyofusefulpostamplificationmanipulationsofthePCR
productwithoutadverselyaffectingyield.Examplesinclude5extensionsthatcontain
restrictionsites,universalprimerbindingsites,orpromotersequences.

Addition of 3' A
nucleotide by Taq
polymerase

TheAmpliTaqandAmpliTaqGoldDNAPolymerases,likemanyotherDNA
polymerases,catalyzetheadditionofasinglenucleotide(usuallyanadenosine)tothe
3endsofthetwostrandsofadoublestrandedDNAfragment.Thisnontemplate
complementaryadditionresultsinadenaturedPCRproductthatisonenucleotide
longerthanthetargetsequence.APCRproductcontainingtheextranucleotideis
referredtoastheplusAform.

Incomplete 3' A nucleotide addition


Because3Anucleotideadditionrarelygoestocompletionwithoutalongextension
stepattheendofthermalcycling(thatis,onlyafractionofthefragmentsreceivethe
extranucleotide),singlebaseladdersoftenform,creatingpeakpatternsthatanalysis
softwaremightnotinterpretcorrectly(Figure6).Theresultingallelecallscanbe
inconsistent,incorrect,ormissingentirely.
Figure6 Split peaks resulting from incomplete 3' A nucleotide addition

DNA Fragment Analysis by Capillary Electrophoresis

33

Chapter2 Experimental Design


Primer design guidelines

Avoiding incomplete 3' A nucleotide addition


Modify
Thermal cycling conditions

Considerations
Increasing the time spent between 60 and 72C promotes 3' A nucleotide addition.
Decreasing the time spent between 60 and 72C inhibits 3' A nucleotide addition.
To use this method effectively, determine the optimal thermal cycling conditions for
each marker in each set of reaction conditions.
Promoting 3' A nucleotide addition has proven to be more successful than removing
3' A. Residual polymerase activity at room temperature (or even at 4C) is often
sufficient to catalyze enough 3' A nucleotide addition to create genotyping problems.
Many protocols increase the final extension step to 30 to 45 minutes to promote 3' A
nucleotide addition.

Magnesium ion concentration

Increasing the magnesium ion concentration promotes 3' A nucleotide addition.


Decreasing the magnesium ion concentration inhibits 3' A nucleotide.
In general, optimizing the magnesium ion concentration is best used in conjunction with
other strategies. If you choose to maximize 3'A nucleotide addition, consider using
AmpliTaq Gold DNA Polymerase at 2.5 mM MgCI2.

Tail
the 5' end of the reverse primer

Brownstein et al. (1996) found that adding additional nucleotides (a tail) to the 5' end
of the reverse PCR primer either promoted or inhibited 3' A nucleotide addition to the
(forward) labeled strand, depending on the sequence of the added nucleotides (Figure7
on page 35).
Magnuson et al. (1996) noticed a correlation between tail sequence and the amount of
3' A nucleotide addition. In particular, they found that adding a single G to the 5' end of
the reverse PCR primer generally resulted in almost complete 3' A nucleotide addition.
Therefore, using a tail to promote 3' A nucleotide addition can consistently yield a
pattern that analysis software can identify.
Reverse-primer tailing has advantages compared to other methods because it:
Works well under diverse reaction conditions
Does not require additional experimental steps
Go to www.lifetechnologies.com for information on ordering tailed primers.

34

DNA Fragment Analysis by Capillary Electrophoresis

Chapter2 Experimental Design


Primer design guidelines

Figure7 Tailed primers and 3' A nucleotide addition

Labeled primer

Without tailed primers


Tailed primer

With tailed primers (plus 7 bp)

Note: Ingeneral,themostreliablestrategyistopromote3Anucleotideadditionby
modifyingthermalcyclingconditionsandMg2+concentration,and(ifnecessary)by
tailingthereverseprimer.

Enzymatic treatment
Ginotetal.(1996)usedT4DNApolymerasetoremovethe3Aoverhangsfrom
treatmentpooledPCRproducts.
Althougheffective,thismethodhasseriouslimitationsbecauseitrequires:
ApostPCRenzymatictreatmentstep
TitratingeachlotofT4DNApolymerasetodetermineoptimalenzyme
concentrationsandtreatmenttimes
IMPORTANT! ExcessT4treatmentcancausePCRproductdegradation.Insufficient
treatmentwillnotremovethe3overhangsandcanmakesomeallelesmoredifficultto
genotype.
Startoptimizationexperimentswith0.5to1unitofT4DNApolymerasein10Lof
pooledPCRproduct.Incubateat37Cfor30minutes.

DNA Fragment Analysis by Capillary Electrophoresis

35

Chapter2 Experimental Design


Dyes

Dyes
IMPORTANT! WerecommendusingonlyThermoFisherScientificdyes.ThermoFisher
Scientificprovidesspectralcalibrationreagentsthathavebeenoptimizedforourdye
sets.
Otherdyes(ormixedisomersofdyes)havevariableemissionspectraandalsorequire
aspectralcalibrationgeneratedforthespecificdyesinusetocorrectforthespectral
overlapbetweenthedyes.Youareresponsibleforobtainingtheappropriatespectral
calibrationreagentsandforoptimizingcustomdyesets.

Dyes and chemical


forms

ThermoFisherScientificdyesareavailableinmultiplechemicalforms.Someformsare
suppliedcoupledtoprimersandothersyoucanusetolabelcustomprimersor
fragments.Eachformhasdistinctadvantagesanddisadvantagesdependinguponthe
intendedapplicationandyourlaboratorysetup.
YoucananalyzephosphoramiditelabeledfragmentswithNHSesterlabeled
fragments,butyoushouldnotcombine[F]dNTPlabeledfragmentswithanyother
labelingmethod.
Table5 Dye chemical forms

Chemical form

Purpose

Available dyes

Post-synthesis 5'-end labeling of oligonucleotides containing a 5'


Aminolink2

NED,

Phosphoramidite
reagents

Preparing custom, 5'-end labeled primers directly on any Thermo


Fisher Scientific DNA synthesizer

6-FAM, HEX, TET,


NED, VIC, PET

[F]dNTPs

Simple internal fluorescent labeling of multiple nucleotides during


PCR amplification

R6G, R110, ROX, TAMRA

Labeled primers in
reagent kits

Microsatellite and human identification applications

5-FAM, JOE,
6-FAM, HEX, TET,
NED, VIC, PET

Labeled size
standard

Generating the sizing curve to size unknown sample fragments

TAMRA, ROX, LIZ

NHS-esters

TAMRA, ROX

SNaPshot Kit dyes: dR110,


dR6G, dTAMRA, dROX

NED, VIC, and PET dye-labeled primers are available only in kits or through the Thermo Fisher Scientific Custom Oligo Service. Contact your
Thermo Fisher Scientific representative or visit our website for information on how to order custom-labeled oligonucleotides.
Matrix standards for spectral calibration available for the 310 instrument only.
For information about synthesizing labeled oligonucleotides, contact your Thermo Fisher Scientific representative.
5-FAM and JOE are available only as labeled primers in certain reagent kits.

Multicomponent
analysis with
fluorescent dyes

Fluorescentdyelabelingenablesyoutoanalyzemultipleindependentmarkers(loci)
inthesamecapillaryinjectionbyusingdifferentdyecolorsinadditiontosizeto
distinguishbetweenmarkers.
Duringdatacollectiononourgeneticanalyzers,thefluorescencesignalsareseparated
bydiffractiongratingaccordingtowavelengthandprojectedontoaCCDcameraina
predictablyspacedpattern.

36

DNA Fragment Analysis by Capillary Electrophoresis

Chapter2 Experimental Design


Dyes

Althougheachdyeemitsitsmaximumfluorescenceatadifferentwavelength,thereis
someoverlapintheemissionspectrabetweenthedyes(Figure8).Tocorrectfor
spectraloverlap,thesoftwareappliesamulticomponentmatrix.Amulticomponent
matrixiscreatedwhenyouperformaspectralcalibrationforadyesetusingamatrix
standard(formoreinformation,seeDyesetsonpage 41andUnderstanding
spectralcalibrationonpage 84).
Figure8 Emission spectra of dyes
Dyes
Normalized Emission

6-FAM

VIC

NED PET

LIZ

100
80
60
40
20
0
500

550

600

650

700

Wavelength (nm)

Factors that affect


dye signal

Fluorescentdyeshavethefollowingcharacteristics:
Emissionspectrum:Theintensityofemittedlight(fluorescence)asafunctionof
thewavelengthoftheemittedlight.
Absorption(excitation)spectrum:Theintensityofemittedlightasafunctionof
thewavelengthoftheexcitinglight.
Absorption(excitation)efficiency:Ameasureoftheprobabilitythatadyewill
absorblightofacertainwavelength,asapercentageoftheprobabilityof
absorptionatthewavelengthofmaximumabsorption.
Quantumyield:Theprobabilitythatitsexcitedstatewillemitaphotonasit
decaysbacktothegroundstate.
Theabilityoftheinstrumenttodetectadyesignaldependsupon:
Theabsorptionefficiencyofthedyeatthewavelengthsoflightemittedbythe
laser
Thelaser/lightsource
Thequantumyieldofthedye
Thedyeconcentration
Theemissionandabsorptionwavelengthsofadyedependupon:
Thechemicalstructureofthedye
Thephysicalenvironment,including:
BufferpHandconcentration
Polymercomposition
WhethertheDNAitisattachedtoissingleordoublestranded
Althoughalteredbythephysicalenvironment,thewavelengthsofmaximumemission
andabsorptionforeachdyealwaysliewithinasmallwavelengthrange.

DNA Fragment Analysis by Capillary Electrophoresis

37

Chapter2 Experimental Design


Dyes

Emission and
absorption
(excitation)
wavelengths and
relative intensities

Themaximumfluorescenceabsorptionandemissionwavelengthsarelistedbelowfor
ThermoFisherScientificNHSesters,dyephosphoramidites,and[F]dNTPbaseddyes.
(Theactualmaximumabsorptionandemissionwavelengthsmaydifferfromthelisted
valuesbecauseoftheinfluenceofthephysicalenvironmentuponthedye.)
Theintensityofemittedfluorescenceisdifferentforeachdye,andyoumustoptimize
sampleconcentrationtoaccountfordifferencesindyesignalstrength.
Examples:
6FAMdyeemitsastrongersignalthanNEDdye.Therefore,togenerate
signalsofequalintensity,youmustloadapproximatelythreetimesasmuch
NEDdyelabeledfragmentsas6FAMdyelabeledfragments.
VICdyeemitsastrongersignalandismorestablethanHEXdye.UseVIC
dyeforweakamplicons.
Table6 Dye Absorption max, emission max, and relative intensities

38

Dye

Absorption Max

Emission Max

Relative
Intensity

5-FAM

494 nm

530 nm

100 RFU

6-FAM

494 nm

522 nm

100 RFU

TET
(310 only)

521 nm

538 nm

100 RFU

VIC

538 nm

554 nm

100 RFU

JOE

528 nm

554 nm

50 RFU

HEX

535 nm

553 nm

50 RFU

LIZ

638 nm

655 nm

50 RFU

NED

546 nm

575 nm

40 RFU

TAMRA

560 nm

583 nm

25 RFU

ROX

587 nm

607 nm

25 RFU

PET

558 nm

595 nm

25 RFU

Intensity (not
to scale)

DNA Fragment Analysis by Capillary Electrophoresis

Chapter2 Experimental Design


Dyes

Points to consider
when selecting
dyes for custom
primers

Whenyouordercustomprimers,youspecifythedyesforlabeling.Basedonthedyes
youspecify,youmustusetheappropriatedyesettoperformaspectralcalibration
(describedinDyesetsonpage 41).
Considerthefollowingwhenselectingdyes:
Onedyeisneededforthesizestandard(redororange).
Using5dyesprovides33%greaterthroughputthanusing4dyes.
UsethemostintensedyesforPCRproductswithlowrecoveryrate(fromlowerto
higherintensity:Blue>Green>Yellow>Red)(seeEmissionandabsorption
(excitation)wavelengthsandrelativeintensitiesonpage 38).
UselessintensedyesforPCRproductwithgoodrecoveryrate.
Selectdyeswithabsorptionmaximathatareasfarapartaspossibletoavoid
overlapandforeasiergenerationofmatrix/spectralcalibration(seeEmission
andabsorption(excitation)wavelengthsandrelativeintensitiesonpage 38).
Considertherelativedyeintensitiesandsampleconcentration(seeEmissionand
absorption(excitation)wavelengthsandrelativeintensitiesonpage 38).

Example: selecting
dyes

Thefollowingfigureshowsthemarkerrange(alleledistribution)andallele
frequenciesforthealligatormicrosatellitelocusAmi8forsamplestakenfrom
Florida/SouthGeorgiaandTexas/Louisiana.
Figure9 Allele distribution for alligator marker Ami-8 in two populations (124 to 156 bp)

Usingahypotheticalsetof10markersasanexample:
Foreachmarker,determinetheexpectedalleledistribution,eitherfrompublished
literatureorfromempiricaltesting.
Determinethedyesthatareappropriatefortherangeofeachmarkerofinterest.

DNA Fragment Analysis by Capillary Electrophoresis

39

Chapter2 Experimental Design


Dyes

Forthishypotheticalsetofmarkers,youmightselect6FAM,VIC,PET,
NEDdyes,andLIZdyeforthesizestandard(seethetablebelow).Thisgroup
ofdyescorrespondstotheG5dyeset,soyouwouldalsoneedtheDS33matrix
standardforspectralcalibration(seeTable7onpage41forthematrixstandard
thatcorrespondstoeachdyeset).
Notethatdifferentdyescanbeusedforsimilarfragmentlengths,andthesame
dyecanbeusedforfragmentsofdifferentlengths.

40

Locus

Marker
Range

Dye

Marker 1

90104

6-FAM

Marker 2

112146

VIC

Marker 3

119177

PET

Marker 4

117202

NED

Marker 5

156190

6-FAM

Marker 6

221253

6-FAM

Marker 7

234282

NED

Marker 8

260342

VIC

Marker 9

311327

6-FAM

Marker10

340380

NED

Capillary electrophoresis array view


of example 10-marker panel

DNA Fragment Analysis by Capillary Electrophoresis

Chapter2 Experimental Design

Dye sets

Dye sets
Dye sets and
matrix standards

Adyesetcorrespondstothegroupofdyesyouselectforlabeling(describedinthe
previoussection).Youusethematrixstandardthatcorrespondstothedyeset(shown
below)toperformaspectralcalibration.Thiscalibrationpreparestheinstrumentfor
detectionofthedyeswithwhichyourprimersarelabeled.Forinformationonspectral
calibration,seeUnderstandingspectralcalibrationonpage 84.
Table7 Dye set and matrix standard components
Dye Set
(ROX, LIZ, and TAMRA dyes are reserved for the size standard)
Dye Set

E5

G5

Matrix
standard

DS-02

DS-30

DS-31

DS-32

DS-33

DS-34

Blue

dR110

6-FAM

6-FAM

5-FAM

6-FAM

6-FAM

Green

dR6G

HEX

VIC

JOE

VIC

TET

Yellow

dTAMRA

NED

NED

NED

NED

HEX

Red

dROX

ROX

ROX

ROX

PET

TAMRA

Orange

LIZ

LIZ

Used on 310 instruments only.


Can be used for custom-labeling primers.
DS-30 versus DS-31: VIC dye emits a stronger signal and is more stable than HEX dye. Use VIC dye for
weak amplicons.

ThekitsavailablefromThermoFisherScientificusethedyesetslistedbelow.
Table8 Dye sets and matrix standards for kits and genotyping applications
Dye Set

Matrix
Standard

SNaPshot Primer Focus, SNaPshot Multiplex

E5

DS-02

Custom oligos

DS-30

Custom oligos, Plant and Microbial AFLP, Bovine and


Canine Stockmarks

DS-31

Stockmarks, AFLP

DS-32

Equine Stockmarks, custom oligos, AmpFlSTR

G5

DS-33

Application

Creating a custom
dye set

IMPORTANT! WerecommendusingonlyThermoFisherScientificdyes.ThermoFisher
Scientificprovidesspectralcalibrationreagentsthathavebeenoptimizedforourdye
sets.
NonThermoFisherScientificdyes(ormixedisomersofdyes)havevariableemission
spectraandalsorequireaspectralcalibrationgeneratedforthespecificdyesinuseto
correctforthespectraloverlapbetweenthedyes.Youareresponsibleforobtainingthe
appropriatespectralcalibrationreagentsandforoptimizingcustomdyesetstoensure
thedyelabelsdonotaffectPCRefficiency.

DNA Fragment Analysis by Capillary Electrophoresis

41

Chapter2 Experimental Design


Size standards

However,the3500Series,3730Series,and3130Seriesinstrumentsdosupportcustom
dyesets.Forinformation,seeChapter4,OptimizingCapillaryElectrophoresison
page67.

Size standards
Functions of a size
standard

Eachunknownsampleismixedwithsizestandardbeforeelectrophoresisandrun
togetherinthesamecapillarywiththesameconditions.Sizestandardsperformtwo
functions:
Allowsizingofsamplepeaks.Asizecurveisgeneratedforeachsample.Because
thesizes(inbp)ofthesizestandardpeaksareknown,thesizesofsamplepeaks
aredeterminedthrougharelativecomparisonofmigrationspeedsduring
electrophoresis.Theuniformspacingofsizestandardfragmentsensuresprecise
sizingthroughoutthesizingrange.
IMPORTANT! Becausethecalledsizeforafragmentcandifferfromitsactualsize,
comparetheallelecallsinsteadofthefragmentsize.
IMPORTANT! Usethesamesizestandard,instruments,andinstrumentconditions
forallsamplesinastudy.Usingdifferentsizestandards,instruments,or
instrumentconditionsmayshiftthesizingofDNAfragments.
Precision,orreproducibility,isthemeasureofinstrumentabilitytogeneratethe
samesizeconsistentlyforagivenfragment.
Formoreinformation,seePreciseversusaccuratesizingonpage 90,
Guidelinesforconsistentsizingonpage 90,andHowtheGeneMapper
Softwareperformssizingonpage 98.
Correctforinjectiontoinjectionvariationsthatresultindifferenceswhen
comparingthesameDNAfragmentsfromdifferentcapillaries,runs,and
instruments.
Whencomparingfragmentsizeacrossinjections,ensurethatdataisanalyzed
withthesamesizingmethodandthesamesizestandarddefinition.

Size-standard peak
intensity

42

Foroptimumperformance,thesignalintensityofthesizestandardpeaksshouldbe
lowerthanorequaltothesignalintensityofthesamplepeaks.Formoreinformation,
seeBalancingsizestandardandsamplepeakintensitiesonpage 77.

DNA Fragment Analysis by Capillary Electrophoresis

Chapter2 Experimental Design


Size standards

Selecting a
GeneScan size
standard

Selectasizestandardwithatleasttwofragmentssmallerandlargerthanyour
unknownsamplefragments,andwithadyethatiscompatiblewiththedyesusedfor
labelingprimers.
LIZ Size Standard, 5-dye chemistry

Expected
marker
length

ROX Size Standard, 4-dye chemistry

GS120
LIZ

GS500
LIZ

GS600
LIZ

GS1200
LIZ

GS350
ROX

GS400HD
ROX

GS500
ROX

GS1000
ROX

(page 47)

(page 50)

(page 45)

(page 47)

(page 49)

(page 49)

(page 50)

(page 51)

120 bp

400 bp

500 bp

600 bp

1000 bp

1200 bp

SNaPshot

Used with
Multiplex Kit.
For denaturing and non-denaturing applications.

Peaks not used for


sizing
Preparing a size
standard

Somesizestandardsincludepeaksthatarenotusedforsizing.Thesepeaksare
denotedwitha*inthefollowingfigures.Thesepeakscanbeusedasanindicatorof
precisionwithinarun.

1. Vortextomixthecontentsofeachsizestandardtubethoroughly,thencentrifuge
brieflytocollecttheliquidatthebottomofthetube.

2. OptimizetheratioofsampletosizestandardandHiDiformamideusingthe
valueslistedbelowasastartingpoint.
Components
Sample
Size standard
Hi-Di

Formamide

3500 Series, 3730 Series, and


3130 Series instruments

310 instrument

0.5 L per reaction

0.5 L per reaction

0.5 L per reaction

0.5 L per reaction

9.0 L per reaction

11.0 L per reaction

Hi-Di Formamide (Part no. 4311320) is purchased separately from the size standard.

3. Createamastermixofthesizestandardandformamide.
4. Addsamplesandmastermixtotubesorwells.
5. Heatthereactionmixfor3to5minutesat95C.Immediatelychillonicefor
2 to 3 minutes,thenloadsamples.
IMPORTANT! Aftersizestandardsaremixedwithformamide,runimmediately.Signal
willdecreasesignificantlyifleftatroomtemperaturefor>1dayorat2to8C
for>5days.Platescanbestoredat20Cforupto1week.

DNA Fragment Analysis by Capillary Electrophoresis

43

Chapter2 Experimental Design


Size standards

GeneScan 120
LIZ Size Standard

Range:15to120bpunderdenaturingconditions
GeneScan 120 LIZ denatured fragment lengths (nt): 9 fragments
15

35

80

20

50

110

25

62

120

Thissinglestrandedsizestandardwasdesignedtoprovideaccuratesizingofshort
DNAfragments.Therefore,itisparticularlyusefulforSNPanalysis.Allfragments
havebeenoptimizedunderawidevarietyofrunconditions.
Figure10 GeneScan 120 Size Standard run under denaturing conditions

GeneScan
500 LIZ Size
Standard

Range:35to500bpunderdenaturingconditions
Thissizestandardisrecommendedforanalysisoftriandtetranucleotide
microsatelliteloci,whichcanoftenexceed400bpinlength.
GeneScan 500 LIZ denatured fragment lengths (nt): 16 fragments
35

139

250

400

50

150

300

450

75

160

340

490

100

200

350

500

Do not use this fragment for sizing. See Peaks not used for sizing on page 43 for information.

OnlyonestrandofthedoublestrandedDNAfragmentsinthissizestandardis
labeled.Theunlabeledstranddoesnotinterferewithpeakdetectionofthelabeled
strandwhenrununderdenaturingconditions.

44

DNA Fragment Analysis by Capillary Electrophoresis

Chapter2 Experimental Design


Size standards

Figure11 GeneScan 500 LIZ Size Standard run under denaturing conditions

GeneScan 600
LIZ and
GeneScan 600
LIZ v2.0 Size
Standards

Note: TheGeneScan600LIZandGeneScan600LIZv2.0SizeStandardscontain
thesamepeaks.TheGeneScan600LIZv2.0SizeStandardcanbeusedfor
normalizationon3500Seriesinstruments.
Range:20to600bpunderdenaturingconditions
GeneScan 600 LIZ denatured fragment lengths (nt): 36 fragments
20

120

220

314

414

514

40

140

240

320

420

520

60

160

250

340

440

540

80

180

260

360

460

560

100

200

280

380

480

580

114

214

300

400

500

600

DNA Fragment Analysis by Capillary Electrophoresis

45

Chapter2 Experimental Design


Size standards

Figure12 GeneScan 600 LIZ Size Standard fragments run under denaturing conditions

Optimizing the 3130 Series instrument run module


Therunmodulesprovidedwiththeseinstrumentsmayneedtobeoptimizedforuse
withtheGeneScan600LIZSizeStandard.Add100secondstotheruntimeif
needed.

Optimizing the 310 instrument run module


Therunmodulesprovidedwiththeseinstrumentshavenotbeenoptimizedforuse
withtheGeneScan600LIZSizeStandard.Add200secondstotheruntimebefore
usingthissizestandard.

46

DNA Fragment Analysis by Capillary Electrophoresis

Chapter2 Experimental Design


Size standards

GeneScan 1200
LIZ Size Standard

Range:20to1200bpunderdenaturingconditions
GeneScan 1200 LIZ Size Standard denatured fragment lengths (nt): 68 fragments
20

280

560

850

30

300

580

860

40

314

600

880

60

320

614

900

80

340

620

920

100

360

640

940

114

380

660

960

120

400

680

980

140

414

700

1000

160

420

714

1020

180

440

720

1040

200

460

740

1060

214

480

760

1080

220

500

780

1100

240

514

800

1120

250

520

820

1160

260

540

840

1200

Thehighfragmentdensity(68fragments)yieldsgreatersizingprecision,and
landmarkfragmentsalloweasypeakpatternidentificationduringdataanalysis.
ThissizestandardisidealforBACfingerprinting,TRFLP,VNTR,STR,andmany
otherDNAfragmentanalysisapplications.
Figure13 GeneScan 1200 LIZ Size Standard run under denaturing conditions

DNA Fragment Analysis by Capillary Electrophoresis

47

Chapter2 Experimental Design


Size standards

Downloading 3130 instrument run modules from our website


Updatedrunmodulesforthe3130SeriesinstrumentandtheGeneScan1200LIZ
SizeStandardareavailableonourwebsite.Beforeusingthedownloadedrunmodules,
adjustasdescribedbelow.Furtheroptimizationmaybenecessary.
36-cm array with POP-4 polymer

50-cm array with POP-4 polymer

Decrease run voltage to 8000 volts

Decrease run voltage to 12,000 volts

Increase run time to 6000 seconds

Increase run time to 6000 seconds

Downloading 3730 instrument run modules from our website


Updatedrunmodulesforthe3730SeriesinstrumentandtheGeneScan1200LIZ
SizeStandardareavailableonourwebsite.Beforeusingthedownloadedrunmodules,
adjustasdescribedbelow.Furtheroptimizationmaybenecessary.
50-cm array
Decrease run voltage to 8000 volts
Increase run time to 6200 seconds

48

DNA Fragment Analysis by Capillary Electrophoresis

Chapter2 Experimental Design


Size standards

GeneScan 350
ROX Size
Standard

Range:35to350bpunderdenaturingconditions
GeneScan ROX 350 denatured fragment lengths (nt): 12 fragments
35

139

250

50

150

300

75

160

340

100

200

350

Do not use this fragment for sizing. See Peaks not used for sizing on page 43 for information.

OnlyonestrandofthedoublestrandedDNAfragmentsinthissizestandardis
labeled.Theunlabeledstranddoesnotinterferewithpeakdetectionofthelabeled
strandwhenrununderdenaturingconditions.

GeneScan 400HD
ROX Size
Standard

340
350

300

200

139

100

150
160

50

35

75

Figure14 GeneScan 350 Size Standard run under denaturing conditions

ThissizestandardusesROXdye.Thehighdensityofmarkerbandsinthisstandard
makesitparticularlyusefulformicrosatelliteanalysis.
Range:50to400bpunderdenaturingconditions
GeneScan ROX 400HD denatured fragment lengths (nt): 21 fragments
50

160

260

360

60

180

280

380

90

190

290

400

100

200

300

120

220

320

150

240

340

OnlyonestrandofthedoublestrandedDNAfragmentsinthissizestandardis
labeled.Theunlabeledstranddoesnotinterferewithpeakdetectionofthelabeled
strand.

DNA Fragment Analysis by Capillary Electrophoresis

49

Chapter2 Experimental Design


Size standards

Figure15 GeneScan 400HD Size Standard run under denaturing conditions

GeneScan
500 ROX Size
Standard

Range:35to500bpunderdenaturingconditions
Thissizestandardisrecommendedforanalysisoftriandtetranucleotide
microsatelliteloci,whichcanoftenexceed400bpinlength.
GeneScan 500 ROX denatured fragment lengths (nt): 16 fragments
35

139

250

400

50

150

300

450

75

160

340

490

100

200

350

500

Do not use this fragment for sizing. See Peaks not used for sizing on page 43 for information.

OnlyonestrandofthedoublestrandedDNAfragmentsinthissizestandardis
labeled.Theunlabeledstranddoesnotinterferewithpeakdetectionofthelabeled
strandwhenrununderdenaturingconditions.

50

DNA Fragment Analysis by Capillary Electrophoresis

Chapter2 Experimental Design


Size standards

Figure16 GeneScan 500 ROX Size Standard run under denaturing conditions

GeneScan 1000
ROX Size
Standard

Range:
100to900bpundernondenaturingconditions
100to539bpunderdenaturingconditions(verifiedwithPOP4polymeronly)
GeneScan 1000 ROX non-denatured fragment lengths (nt): 17 fragments
47

93

292

695

51

99

317

946

55

126

439

82

136

557

85

262

692

If run under denaturing conditions (Figure17 on page 52), fragments run 18 nucleotides shorter than the
lengths listed above and some or all of the peaks appear split.
Do not use this fragment for sizing. See Peaks not used for sizing on page 43 for information.

BothstrandsoftheGeneScan1000ROXSizeStandardfragmentsarelabeledand
areusedfornondenaturingapplications.

DNA Fragment Analysis by Capillary Electrophoresis

51

Chapter2 Experimental Design


Ordering custom primers

Figure17 GeneScan 1000 ROX Size Standard run under denaturing conditions. Fragments
run 18 nt shorter than lengths obtained under non-denaturing conditions (see the table on the
previous page). Under denaturing conditions, sizing is not accurate above 539 nt, therefore the
and 674-nt (corresponds to the 692-nt) peak is not shown in the figure.

Note: Underdenaturingconditions,thetwostrandsofthisdoublylabeled
sizestandardmigrateatdifferentrates,appearingassplitpeaks.Toensuresizing
precisionandareliablesizestandarddefinition,youmustdefineonepeakfromeach
splitpeakpairinthesizestandarddefinition.

Ordering custom primers


Youcanobtaincustom5endlabeledprimersfromtheThermoFisherScientific
CustomOligoService.Forinformation,seeourwebsite.
Orderlabeledandunlabeledprimerpairsforthemarkersofinterest.

Testing the primers and optimizing conditions with test DNA panel
Testing

Beforeusingprimersinananalysis,testtheprimersandoptimizesamplepreparation,
PCR,andelectrophoresisconditions.
CreateapaneloftestDNAsamplestoensurethatexpectedallelesaredetectedfor
eachmarker.UseDNAsamplesthatarerepresentativeofyouroverallstudytocapture
asmuchallelicvariationaspossible.CEPHIndividual134702ControlDNAis
availablefromThermoFisherScientificandcanbeusedinyourtestDNApanel.
TestDNApanelguidelines:
Include8to16samples
Usesamplesofgoodqualitythatarewellquantified
UseequalconcentrationsofDNAsamples
Note: Ifoptimizationofsignalintensityisnecessaryforagivensample,injectthe
samplemultipletimesusingarangeofinjectionparameters.

52

DNA Fragment Analysis by Capillary Electrophoresis

Chapter2 Experimental Design


Testing the primers and optimizing conditions with test DNA panel

Orderunlabeledprimersforthemarkersofinterestandoptimizeamplification
conditionsonyourDNAtestpanel.Youmayneedtooptimizeavarietyofparameters
includingannealingtemperature,andvariablessuchasmagnesiumconcentrationand
primerconcentrationtoensurethattheprimersworkunderuniversalconditions.
Bandsarevisualizedonagarosegelswithethidiumbromidestaining.

Optimizing
conditions

Theintensityofemittedfluorescenceisdifferentforeachdye,andyoumustoptimize
sampleconcentrationtoaccountfordifferencesindyesignalstrength.Forexample,to
generatesignalsofequalintensity,youmustloadapproximatelythreetimesasmuch
NEDdyelabeledfragmentsas6FAMdyelabeledfragments.
Formoreinformation,see:
Chapter3,OptimizingPCRonpage55
Chapter4,OptimizingCapillaryElectrophoresisonpage67

DNA Fragment Analysis by Capillary Electrophoresis

53

54

Chapter2 Experimental Design


Testing the primers and optimizing conditions with test DNA panel

DNA Fragment Analysis by Capillary Electrophoresis

Optimizing PCR

ThischaptercontainsgeneralinformationforPCR.Forapplicationspecific
informationonPCR,seetheapplicationchapterslaterinthisguide.
Thischaptercovers:

Safetyinformation .................................................... 55

Isolating,purifying,quantifying,andstoringDNA ........................ 55

Handlingprimers ..................................................... 57

UsingcontrolDNA.................................................... 57

Reactionvolumesandplatetypes....................................... 58

Reagentconcentrations................................................ 59

Preventingcompetingsidereactions:hotstartPCR........................ 60

ThermalcyclingparametersVeritiThermalCyclers...................... 60

Thermalcyclingparameters9700ThermalCyclers ........................ 62

Optimizingthermalcyclingparameters .................................. 63

Avoidingcontamination ............................................... 64

Safety information
IMPORTANT! Foreverychemical,readtheSafetyDataSheets(SDSs)andfollowthe
handlinginstructions.Wearappropriateprotectiveeyewear,clothing,andgloves.
Note: FortheSDSsofchemicalsnotdistributedbyThermoFisherScientific,contact
thechemicalmanufacturer.

Isolating, purifying, quantifying, and storing DNA


Isolating DNA

DNAisolationmethodsdependonyourstartingDNAsource.Refertoguidelinesfor
yourapplicationforinformationonisolatingDNA.
IMPORTANT! DONOTFREEZEBLOODSAMPLESbeforeDNAisolation.Freezing
canlyseredbloodcells,andincreasetheconcentrationofPCRinhibitorsinDNA
samples.

DNA Fragment Analysis by Capillary Electrophoresis

55

Chapter3 Optimizing PCR


Isolating, purifying, quantifying, and storing DNA

Purifying DNA

Thequality,accuracy,andamplifiedlengthofaDNAfragmentcanbesignificantly
affectedbycharacteristicsofthesampleitselfandthemethodusedforpurification.
IMPORTANT! ThesuccessofAFLPanalysisisparticularlydependentuponthe
qualityofDNA.
Selectamethodbasedonthesamplesourceortissuetype,howitwasobtainedfrom
itssource,andhowitwashandledorstoredbeforepurification.Goto
www.lifetechnologies.comforthelatestinformationonDNApurification.

Quantifying DNA

ForPCRwithcustomprimers,optimizeDNAconcentrationforyourapplication.
Concentrationmayrangefrom10to100ngofpurifiedDNAperreaction.
ItisalmostalwaysnecessarytodilutePCRamplificationproductsbeforeaddingthem
tothesampletube.Typically,therequireddilutionis1:3to1:80(PCRproductto
distilled,deionizedwaterorHiDiFormamide).
BeginbyoptimizingPCRrunconditionsforyourspecificapplication.Thenruna
dilutionseriesonyourinstrumenttodeterminetheoptimaldilution.Alternatively,
run1LofPCRproductonaminigel.If,afterethidiumbromidestaining,theproduct
signalisvisiblebutnotoversaturated,trya1:10dilution.
Afterdeterminingtheoptimaldilutionratio,youcanusethesamedilutionsfor
subsequentanalysesbecausePCRyieldsshouldbefairlyconsistent.Anychangesto
thePCRconditionsortheprimerdesignmayrequiredifferentdilutions.
Note: Differentdyesemitdifferentfluorescentintensities.Therefore,PCRproduct
concentrationsmayneedoptimizationdependingonrelativefluorescenceintensity
duringelectrophoresis.SeeEmissionandabsorption(excitation)wavelengthsand
relativeintensitiesonpage38.

Storing prepared
DNA before or after
PCR

56

Storethepreparedsamplesat20Cto4Cuntilyouperformcapillary
electrophoresis.

DNA Fragment Analysis by Capillary Electrophoresis

Chapter3 Optimizing PCR


Handling primers

Handling primers
Reconstituting and
diluting primers

Primersarecommonlyshippedinalyophilizedstate.Theunitsofalyophilizedprimer
aregivenasamass,inpicomoles.
Tocreateastockofprimersorprobe,reconstitutetheprimerorprobeinsterile
1 TE buffer(1mMTris,0.1mMEDTA,pH8.0)orsterile,nucleasefreewater.

Quantifying
primers
Storing primers

Measuretheprimerquantitywithaspectrophotometerusingaprimerspecific
absorptioncoefficient.
20Cto80Cforstocksolution(undiluted,keepconcentrationashighas
possible)
+4Cforworkingsolution,dilutedappropriately(uptoonemonth)

Using control DNA


Purpose of control
DNA

ServesasapositivecontrolfortroubleshootingPCRamplification
ControlDNAallowsyoutodistinguishbetweenproblemswiththesampleDNA
(thecontrolDNAamplifiesbutsamplesdonot)andproblemswithreagents,
thermalcyclers,orprotocols(thecontrolDNAdoesnotamplify).
Allowsyoutomonitorsizingprecision
BecausethecontrolDNAisnotusedtocalculatethesizingcurve,youcanusethe
sizesobtainedduringdifferentcapillaryinjectionstoverifythatsizingprecision
(reproducibility)iswithinacceptablelimits.
Allowsyoutocorrelatethefragmentsizesthatareobtainedindifferentrunsor
ondifferentinstruments.

Guidelines for use

AmplifyatleastonecontrolDNAsampleineveryPCRrun.
IncludeatleastoneinjectionofamplifiedcontrolDNAduringeveryseriesof
capillaryruns.Useonecontrolinjectionforeveryvariationintheelectrophoresis
parameters.

CEPH 1347-02
Control DNA

CEPHIndividual134702ControlDNAisavailableforhumanstudiesfromThermo
FisherScientific(Partno.403062).

DNA Fragment Analysis by Capillary Electrophoresis

57

Chapter3 Optimizing PCR


Reaction volumes and plate types

Reaction volumes and plate types


Reaction volumes

ReactionvolumesforThermoFisherScientificPCRthermalcyclersare5to100 L.

Using small
amounts of
template

Althoughreactiontubesusuallydonotneedtobesterilizedorsiliconized,use
autoclavedtubeswhenamplifyingwithquantities(approximately150to500pg)of
startingDNAtemplate.
AutoclavedPCRtubesareavailablefromThermoFisherScientific(seeThermal
cyclersandaccessoriesonpage193).

Plate types
Thermal Cycler
Veriti 96-Well
Thermal Cycler

Table9 Reaction plates for each thermal cycler


Block Format

Reaction Plate Type

Networking
capability

Uses

0.1 mL or 0.2 mL
Alloy VeriFlex
Blocks

Standard 0.2 mL and


Fast 0.1 mL 96-well
formats

Yes

Veriti 384-Well
Thermal Cycler

0.02 mL aluminum
single block

384-well plate

Yes

5 to 20 L high throughput,
small sample volume.

Dual 96-Well
GeneAmp PCR
System 9700

2 aluminum
0.2 mL 96-well
blocks

96-well, 0.2 mL format

No

10 to 100 L high throughput,


small sample volume.

Dual 384-Well
GeneAmp PCR
System 9700

2 aluminum
0.02 mL 384-well
blocks

384-well, 0.02 mL format

No

5 to 20 L high throughput,
small sample volume.

Auto-Lid Dual
384- Well
GeneAmp PCR
System 9700

2 aluminum
0.02 mL 384-well
blocks

384-well plate

No

5 to 20 L high throughput,
small sample volume with
robotic capability.

2720 Thermal
Cycler

0.2 mL aluminum
single block

96-well, 0.2 mL format

No

Ideal for basic PCR using


0.2 mL reaction tubes or
96-well reaction plates.

58

10 to 80 L medium/high
throughput.
VeriFlex Blocks provide
better than gradient PCR
optimization.

DNA Fragment Analysis by Capillary Electrophoresis

Chapter3 Optimizing PCR


Reagent concentrations

Reagent concentrations
ThefollowingfactorscanaffectoverallyieldofspecificDNAtargetsequences:
dNTPconcentration
Magnesiumionconcentration
Primerconcentration
Templateconcentration
Enzymeconcentration

dNTP
concentration

InthestandardGeneAmpPCRprotocol,theconcentrationofeachdeoxynucleoside
triphosphate(dNTP)is200M.
Inmostcases,lowerdNTPconcentrationsdonotsignificantlyaffecttheyieldofPCR
amplificationproductandwillincreasethefidelityofthePCRamplificationproduct.
However,forefficientbaseincorporation,keepthefourdNTPconcentrationsbalanced
andabovetheestimatedKmofeachdNTP(10to15M).
SomeapplicationsmightrequirehigherdNTPconcentration(especiallywhendNTP
analoguesareused).However,excessdNTPsdecreaseenzymefidelity.

Magnesium ion

DNApolymerasesrequirefreemagnesiumioninsolutionforactivity.FormostPCR
amplifications,youcanrelateproductyieldandspecificityaswellasenzymefidelity
tothefreemagnesiumion(Mg2+)concentration:
[freeMg2+]=[totalMg2+][totaldNTP]2[EDTA]
Ingeneral,anincreaseinfreemagnesiumconcentrationleadstoanincreaseinproduct
yieldbutadecreaseinspecificityandfidelity.Toidentifythemagnesium
concentrationthatgivesthebestcompromisebetweenyieldandspecificity,inthe
presenceof800MtotaldNTPconcentration,runaMgCI2reactionseriesin50M
incrementsovertherangefrom100to400MMgCI2.

Template
concentration

TheconcentrationoftemplateinasamplecanaffectthesuccessofPCRamplification.
Toomuchtemplatepromotesnonspecificbindingofprimerstosecondarysitesor
changesthepHofthereactionmix.Toolittletemplatecanresultinpooryields,
especiallyifthetemplateisdegraded.
Evenverylowtemplateconcentrations(10copies)areoftensufficientforsuccessful
PCRamplification.
IfthestartingsampleisDNA,youcanuseupto20,000copiesofthetargettostart
optimizationexperiments.Ingeneral,thistranslatesto:
1to5ngofclonedtemplate
200pgto1ngofgenomicDNA
StartoptimizationexperimentswithlessgenomicDNAifstartingsampleislimited.
Withclean,goodqualitygenomicDNA,500to1000pgofstartingsampleistypically
sufficient.

DNA Fragment Analysis by Capillary Electrophoresis

59

Chapter3 Optimizing PCR


Preventing competing side reactions: hot-start PCR

Enzyme
concentration

FormostPCRapplications,2.0to2.5unitsofAmpliTaqGoldDNAPolymeraseis
recommendedforeach100Lreactionvolume.
Note: Toavoidtheinaccuraciesinvolvedinpipetting0.5Lamountsofenzymeinto
eachreaction,prepareafreshmastermixofreagentsandaddtheenzyme.

Preventing competing side reactions: hot-start PCR


When to use

Considerusingthehotstarttechniquewheneveryouneedtoimprovethespecificity
andsensitivityofyourPCRamplifications.Lossofspecificityandsensitivityareoften
causedbycompetingsidereactions,whichusuallyoccurduringtheprePCRsetup
period.(Acommoncompetingsidereactioninvolvestheamplificationofnontarget
sequencesinbackgroundDNAduetomisprimingortoprimeroligomerization.)

Limitations and
alternatives

Thehotstarttechniqueiscumbersome.Ifyouhavehighthroughputneeds,switching
toAmpliTaqGoldDNAPolymerasewillgivethesamebenefitsasperformingthe
hotstarttechnique,withouttheneedforusingwaxbarriersoropeningreactiontubes.
IfyouarealreadyusingAmpliTaqGoldDNAPolymerase,performingthehotstart
techniquewillnotimprovethespecificityandsensitivityofPCRamplification.
Componentsnecessaryforamplificationmustbekeptseparatelysothatcritical
reactantsdonotmixuntilreachingatemperaturesufficientlyhightosuppressprimer
selfannealingorannealingtonontargetsequences.
Note: AlthoughmanualhotstartPCRcanincreasespecificityandyield,itis
inconvenientandyoucanencounterreproducibilityandcontaminationproblems.

Thermal cycling parameters Veriti Thermal Cyclers


AmpliTaq_Gold

60

UsethisprofileforhotstartPCRinplaceoflaborintensivemethodssuchasmanual
hotstartorwaxbeadmediatedhotstarttechniques.TheHotstarttechniquehelpsto
minimizetheformationofprimerdimersornonspecificproducts,therebyincreasing
specificityandsensitivityofPCR.ThisprofilespecifiesaprePCRheatstepfor
activationofAmpliTaqGoldDNAPolymerase.

DNA Fragment Analysis by Capillary Electrophoresis

Chapter3 Optimizing PCR


Thermal cycling parameters Veriti Thermal Cyclers

General PCR

UsethisprofileforstandardPCR.

Time-release PCR

UsethisprofilewithAmpliTaqGoldDNAPolymerase.Thismethodminimizesthe
prePCRactivationstepandaddsaminimumof10additionalcycles,allowingfor
slowactivationoftheenzymeduringcycling.Thisprovidesasimplemethodwhere
polymeraseactivityincreasesmoreslowlyasproductaccumulates,improving
specificity.

Touchdown PCR

Usethisprofileiftheoptimalannealingtemperatureisnotknown.Thismethod
incrementallydecreasestheannealingtemperatureinearlycyclestomaximizethe
yieldofspecificproducts.

DNA Fragment Analysis by Capillary Electrophoresis

61

Chapter3 Optimizing PCR

Thermal cycling parameters 9700 Thermal Cyclers

Thermal cycling parameters 9700 Thermal Cyclers


AmpliTaq_Gold

UsethisprofileforhotstartPCRinplaceoflaborintensivemethodssuchasmanual
hotstartorwaxbeadmediatedhotstarttechniques.Thehotstarttechniquehelpsto
minimizetheformationofprimerdimersornonspecificproducts,therebyincreasing
specificityandsensitivityofPCR.ThisprofilespecifiesaprePCRheatstepfor
activationofAmpliTaqGoldDNAPolymerase.
1 Hld

3 Tmp 35 Cycles

95.0
5:00

95.0
0:15

55.0
0:15

4.0

Method: AmpliTaq Gold

Start
F1

General PCR

2 Holds

72.0 72.0
0:30 7:00

F2

F3

Return

F4

F5

UsethisprofileforstandardPCR.
1 Hld 3 Tmp 35 Cycles 2 Holds
95.0
1.00

95.0
0:15

Start

95.0 94.0
12:00 0:15
55.0
0:15

Start

F3

72.0
0:30

3 Tmp 20 Cycles
89.0
0:15

55.0
0:15

Method: LMS2
F2

Time Release PCR

F3

F4

Return

F4

F5

72.0
0:30

3 Tmp x 10
72.0
0:30

55.0
0:15

Return

Start

F5

F1

3 Tmp 20 Cycles 2 Holds


89.0
0:15

55.0
0:15

72.0 72.0
0:30 10:00

Return

Method: LMS2
F2

F3

4.0

F4

F5

UsethisprofilewithAmpliTaqGoldDNAPolymerase.Thismethodminimizesthe
prePCRactivationstepandaddsaminimumof10additionalcycles,allowingfor
slowactivationoftheenzymeduringcycling.Thisprovidesasimplemethodwhere
polymeraseactivityincreasesmoreslowlyasproductaccumulates,improving
specificity.
1 Hld 3 Tmp 40 Cycles 2 Holds
95.0
1.00

Start
F1

62

F2

UsethisprofilewithLinkageMappingSetprimers.LinkageMappingSetprimersare
foranalysisofselectmicrosatellitelocifromtheGnthonhumanlinkagemap.

1 Hld 3 Tmp 10 Cycles

F1

4.0

Method: General PCR

F1

LMS2

55.0 72.0 72.0


0:15 0:30 7:00

95.0
0:15

55.0 72.0 72.0


0:15 0:30 7:00

4.0

Method: Time Release PCR Return


F2

F3

F4

F5

DNA Fragment Analysis by Capillary Electrophoresis

Chapter3 Optimizing PCR


Optimizing thermal cycling parameters

Touchdown PCR

Usethisprofileiftheoptimalannealingtemperatureisnotknown.Thismethod
incrementallydecreasestheannealingtemperatureinearlycyclestomaximizethe
yieldofspecificproducts.
2 Tmp x 20
94.0
0:15

2 Tmp x 10
94.0
0:15

65.0
0:30
*

Start
F1

XL PCR

55.0
0:30

Method: Touchdown PCR


F2

F3

Return

F4

F5

Usethisprofileforamplificationof5to40kbPCRproductsusingrTthDNA
Polymerase,XLanduniquereactionconditions.Byprovidinglongertemplates,XL
PCRcomplementstechnologiesforrapid,longrangePCR.Morecompletegenescan
beamplifiedinonereactionfromknownexpressedsequences,allowingmoreintrons
tobespanned.YoucanuseXLPCRtoamplifythecontroltarget(a20.8kbproduct
fromLambdaDNA)suppliedinthekit.
1 Hld 2 Tmp X 16 2 Tmp X 12
94.0 94.0
1:00 0:15

68.0
10:00

Start
F1

94.0
0:15

2 Holds

72.0
68.0 10.00
10.00
*

Return

Method: XL PCR
F2

F3

4.0

F4

F5

Optimizing thermal cycling parameters


Optimizing
temperature

SixindependenttemperatureblocksareavailablefortheVeritiThermalCycler.Each
blockprovidesprecisecontroloverthermalcyclingparameteroptimization.For
information,refertoourwebsite.
Tofindtheoptimalthermalcyclingparameters,performaseriesofrunsvaryingthe
annealingordenaturationtemperaturesin2Cincrements.
Note: Donotvarymorethanoneparameteratatime.
Annealing temperature
change

Positive effects

Negative effects

Decreased

Increased PCR product yield

Increased amplification of
non-specific products
(background)

Increased

Increased PCR specificity

Reduced PCR yield

DNA Fragment Analysis by Capillary Electrophoresis

63

Chapter3 Optimizing PCR


Avoiding contamination

Guidelines

Thefollowingtablesummarizestheeffectsofmodifyingtemperaturecontrol
parametersonPCRperformance.
Change in thermal cycling parameter
Increase denaturation temperatures
(up to 96C)

Effect on PCR performance


Can be necessary to allow denaturation,
especially with G+C-rich templates
Can also cause template degradation by
depurination

Decrease annealing temperatures

Can increase yield, but can reduce specificity

Increase annealing temperatures

Increases specificity, but can reduce yield

Set the denaturation, annealing, and


extension step to at least:

Allows samples to reach thermal equilibrium


at each stage

15 seconds (preferably 30 seconds) with


the GeneAmp PCR System 9700
45 seconds using thin-walled tubes with
the DNA Thermal Cycler
Use the autoextension (or AutoX) function
of a thermal cycler to allow longer
extension times in later cycles

Increases yield by allowing complete extension


of PCR product in later cycles

For most applications, an extension temperature of 72C is effective and rarely requires optimization. In the
two-temperature PCR process, the combined annealing/extension step temperature should range from
60 to 70C.

Avoiding contamination
PCRprotocolsareextremelysensitivetocontaminantsintheDNA.Althoughmany
protocolsdescribesimpleorfastextractionorpurificationmethods,carefully
evaluateanychangesorimprovementsinextractionorpurificationmethods.(Also,be
surethatthephysicalandchemicalconditionofthesampleitselfareadequateforthe
intendedlabelingandassaymethods.)

PCR setup work


area

IMPORTANT! TheseitemsshouldneverleavethePCRSetupWorkArea.
Calculator
Gloves,disposable
Markerpen,permanent
Microcentrifuge
Microcentrifugetubes,1.5mL,or2.0mL,orotherappropriatecleantube(for
MasterMixpreparation)
Microcentrifugetuberack
Pipettetips,sterile,disposablehydrophobicfilterplugged
Pipettors
Tubedecapper,autoclavable
Vortex

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DNA Fragment Analysis by Capillary Electrophoresis

Chapter3 Optimizing PCR


Avoiding contamination

Amplified DNA
work area

IMPORTANT! PlacethethermalcyclersintheAmplifiedDNAWorkArea.
Youcanusethefollowingsystems:
Veriti96WellThermalCycler
GeneAmpPCRSystem9700withtheSilver96WellBlock
GeneAmpPCRSystem9700withtheGoldplatedSilver96WellBlock

Avoiding
contamination
from the
environment

Toavoidgeneralcontamination,takethefollowingprecautionarymeasures:
Changepipettipsbetweensamples.
Usefilterpluggedpipettips.
Cleananyworkcontaminatedsurfaceusingaclothsoakedwith50%bleach.
IMPORTANT! Beforecleaningthesampleblockofathermalcycler,refertothe
instrumentuserguidefortheproperprocedure.
Closesampletubeswhennotusingthem.
AlwaysrunanoDNAnegativecontrol.
AnegativecontrolcontainsnotemplateDNA,onlyprimersandtheDNAdiluent
(usuallywaterorbuffer).
Aliquotreactionreagentstominimizethenumberoftimesyouuseastock
solution.

Avoiding PCR
product carryover

PCRproductcarryoveristhecontaminationofanunamplifiedsamplewithpreviously
amplifiedDNA.

Why carryover is a particular concern


PCRproductcarryoverisaparticularconcernbecauseamplifiedPCRproductisan
idealtemplateforsubsequentamplificationsofthatsametarget.
AsinglePCRamplificationproducesalargenumberofcopies(asmanyas1013).The
inadvertenttransferofevenatinyvolumeoraerosolofamplifiedproductcan
significantlycontaminateunamplifiedsamples.Contaminationcanresultin
falsepositivesandthedetectionandamplificationofthecontaminatingsequence
insteadofthetargetsequence.

Precautionary measures
Usepositivedisplacementpipettesorfilterpluggedpipettetips.
Physicallyseparatereactionsbeforeandafteramplification.
HandlepreandpostPCRsolutionswithseparatesetsof:
Pipettes
Pipettetips
Microcentrifugetubes
Gloves
UseAmpEraseUNGinreactionmixturestopreventthesubsequent
reamplificationofdUcontainingPCRproducts.

DNA Fragment Analysis by Capillary Electrophoresis

65

Chapter3 Optimizing PCR


Avoiding contamination

For more
information

66

ThermoFisherScientificsuppliestheGeneAmpPCRCarryoverPreventionKit
(Part no. N8080068)andAmpEraseUNG(Partno.N8080096)toensurethatPCR
productsarenotreamplifiedinsubsequentPCRamplifications.

DNA Fragment Analysis by Capillary Electrophoresis

Optimizing Capillary
Electrophoresis

Safetyinformation .................................................... 68

Overview............................................................ 68

3500Seriesinstruments ................................................ 69

3730Seriesinstruments ................................................ 73

3130Seriesinstruments ................................................ 75

310instruments....................................................... 76

Optimizingsampleloadingconcentration ................................ 76

Optimizingsignalintensity............................................. 77

Optimizingelectrokineticinjectionparameters ............................ 78

Optimizingelectrophoresisconditions ................................... 80

Otherfactorsthataffectelectrophoresis.................................. 82

Understandingspatialcalibration....................................... 84

Understandingspectralcalibration...................................... 84

DNA Fragment Analysis by Capillary Electrophoresis

67

Chapter4 Optimizing Capillary Electrophoresis


Safety information

Safety information
Forsafetyandbiohazardguidelines,refertotheSafetysectionintheuserguidefor
yourinstrument.Foreverychemical,readtheSafetyDataSheets(SDSs)andfollowthe
handlinginstructions.Wearappropriateprotectiveeyewear,clothing,andgloves.
Note: FortheSDSsofchemicalsnotdistributedbyThermoFisherScientific,contact
thechemicalmanufacturer.

Overview
Note: Performcapillaryelectrophoresisunderwellcontrolledconditionswith
standardoperatingprocedures.Werecommendusingadedicatedinstrument
platformforanexperimenttominimizerandomerrorduetosizingimprecision.
Thischaptercontainsgeneralinformationforcapillaryelectrophoresis.For
applicationspecificinformationoncapillaryelectrophoresis,seetheapplication
chapterslaterinthisguide.

Thermo Fisher
Scientific Genetic
Analyzers
Table10 Thermo Fisher Scientific Genetic Analyzers (capillary electrophoresis technology)

Instrument

Number of
capillaries

3500

3500xL

24

3730

48

3730xl

96

3130

3130xl

16

310

Capillary
array
length
36 and
50 cm

Polymer
type
POP-7
POP-4
POP-6

Sample capacity
96-well plates and
8-tube strips

Data Collection Software


version
3500 Series Software v1.0
or later

96- and 384-well plates


and 8-tube strips
36 and
50 cm

POP-7
POP-6

96- and 384-well plates

Data Collection Software


v3.0 or v3.1 or later

22, 36, and


50 cm

POP-7
POP-4
POP-6

96- and 384-well plates

Data Collection Software


v3.0 or v3.1 or later

47 cm

POP-4
POP-6

Up to 48 or 96 sample
tubes

310 Data Collection


Software

3500 Series instruments: 36-cm capillary arrays and POP-4 polymer are used for HID applications only.

Formoreinformationoninstruments,seeInstrumentdocumentationonpage199.

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DNA Fragment Analysis by Capillary Electrophoresis

Chapter4 Optimizing Capillary Electrophoresis


3500 Series instruments

Overview of run
modules

A run module contains electrophoresis parameters, such as oven temperature, detector


cell temperature, ramp rate, injection time and voltage, and run time or run voltage.
Each run module provided with the Data Collection Software has been optimized for a
specific instrument, polymer, capillary, and sample configuration (run modules for the
3500 Series instrument are embedded in the instrument protocols). Some settings, such
as injection time/voltage or run time, may need to be optimized for your instrument
and application.
Updated versions of run modules for your instrument may be available on our
web site.

Using controls

To simplify troubleshooting, run controls with every run for multicapillary


instruments or each set of runs on 310 instruments.

3500 Series instruments


The 36-cm capillary arrays and POP-4 polymer are used for HID applications only.

Run modules

Table11 3500 Series instrument run modules


Configuration

23 hours Throughput

Performance

Run modules type


and
run modules name

Fragment analysis

Sizing Precision
Capillary
length

Polymer

Run
Time

3500

3500xL

Range

50 cm

POP-7

40 min

280

840

40 to
520

<0.15

<0.30

NA

50 cm

POP-6

100 min 112

336

20 to
550

<0.15

<0.30

NA

50 cm

POP-7

125 min

88

360

40 to
700

<0.15

<0.30

<0.45

36 cm

POP-4

35 min

312

936

60 to
400

<0.15

NA

NA

36 cm

POP-7

26 min

424

1272

60 to
400

<0.15

NA

NA

50 cm

POP-7

30 min

376

1104

40 to
120

<0.50

NA

NA

FragmentAnalysis50_POP7
Fragment analysis
FragmentAnalysis50_POP6
Long fragment analysis
LongFragAnalysis50_POP7
HID
HID36_POP4
HID
HID36_POP7
SNaPshot
SNaPshot50_POP7

50 bp- 401 bp- 601 bp400 bp 600 bp 1200 bp

Throughput (samples/day): The total number of samples run in 23 hours (0.5 hour for user interaction and 0.5 hours for warm-up time).
Resolution range: The range of bases over which the resolution (peak spacing interval divided by the peak width at half-max in a GeneScan
600 LIZ or GeneScan 1200 LIZ Size Standard sample sized with a third order fit) is 1. The table shows the resolution range in 90% of
samples.
Sizing precision: Standard deviation of sizes for one allele in the DS-33 install standard sized with the GeneScan 600 LIZ Size Standard v2.0
across multiple capillaries in the same run. For one injection to pass, 100% of the alleles in that injection must meet the intra-run sizing
precision specifications. The table shows the sizing precision of 100% of alleles in 90% of samples.
Not applicable because of the size of the fragments collected in the run.

DNA Fragment Analysis by Capillary Electrophoresis

69

Chapter4 Optimizing Capillary Electrophoresis


3500 Series instruments

Performance

Table12 3500 Series instrument resolution, largest fragment, and sizing


Sizing Precision of 100% of Alleles
in 90% of Samples

General

Multirun Sizing of 100% of Alleles in


90% of Samples

Resolution
Range in 90%
of Samples

Largest
Fragment
Collected in
90% of
Samples

50400 bp

401600
bp

6011,200
bp

50400 bp

401600
bp

6011,200
bp

40 to 520

600

<0.15

<0.30

NA

<1 bp

<2 bp

NA

20 to 550

600

<0.15

<0.30

NA

<1 bp

<2 bp

NA

40 to 700

1,200

<0.15

<0.30

<1 bp

<2 bp

<3 bp

60 to 400

420

<0.15

NA

NA

<1 bp

NA

NA

60 to 400

420

<0.15

NA

NA

<1 bp

NA

NA

40 to 120

120

<0.50

NA

NA

<1 bp

NA

NA

Dye sets and


matrix standards

Table18onpage86liststhedyesetsandmatrixstandardseachinstrument.

Creating a custom
dye set

IMPORTANT! WerecommendusingonlyThermoFisherScientificdyes.ThermoFisher
Scientificprovidescalibrationreagentsthathavebeenoptimizedforourdyesets.
NonThermoFisherScientificdyes(ormixedisomersofdyes)havevariableemission
spectraandrequireaspectralcalibrationgeneratedforthespecificdyestocorrectfor
thespectraloverlapbetweenthedyes.Youareresponsibleforobtainingthe
appropriatespectralcalibrationreagentsandforoptimizingcustomdyesetstoensure
thedyelabelsdonotaffectPCRefficiency.

1. IntheDyeSetlibrary,clickCreate.
2. Enteradyesetname.
3. SelectachemistryandtheAnyDyedyesettemplate.
4. Selectthedyecolorstouseandsetthecalibrationpeakorder:

70

DNA Fragment Analysis by Capillary Electrophoresis

Chapter4 Optimizing Capillary Electrophoresis


3500 Series instruments

a. Selectthedyecolorstouse,whichspecifiestheordernumberofthedyeused
internallybythesoftware.Notethatwhenyoudeselectadye,theorder
numberofthedyeusedinternallybythesoftwarechanges.Theexamples
belowarefora3500Seriesinstrumentwith6dyesupport,butthelogic
appliesto4and5dyes.
InExample1withalldyesselected,internalordernumberisBlue(1),
Green(2),Yellow(3),Red(4),Purple(5),Orange(6).
InExample2withthePurpledyedeselected,internalordernumberis
Blue(1),Green(2),Yellow(3),Red(4),Orange(5)theinternalorder
numberofOrangechangesto5.
InExample3withtheBlue,Yellow,andPurpledyesdeselected,internal
ordernumberisGreen(1),Red(2),Orange(3)theinternalorder
numberofGreenchangesto1,Redchangesto2,andOrange
changes to 3.

b. Specifytheorderofthepeaksinthecalibrationstandardyouareusing.Use
theinternalordernumberofthedyebasedonthedyesselected.
IMPORTANT! TheCalibrationPeakOrderfieldsdonotcorrespondtothedye
colorsdisplayedabovetheCalibrationPeakOrderfields.
InExample1onthenextpage,iftheorderofthepeaksinthe
calibrationstandardyouareusingisOrange,Red,Yellow,Blue,Green,
Purple,specifyforCalibrationPeakOrder:6(Orange),4(Red),
3 (Yellow),1(Blue),2(Green),5(Purple).
InExample2iftheorderofthepeaksinthecalibrationstandardyou
areusingisOrange,Red,Yellow,Blue,Green,specifyforCalibration
PeakOrder:5(Orange),4(Red),3(Yellow),1(Blue),2(Green).

DNA Fragment Analysis by Capillary Electrophoresis

71

Chapter4 Optimizing Capillary Electrophoresis


3500 Series instruments

InExample3iftheorderofthepeaksinthecalibrationstandardyou
areusingisOrange,Red,Green,specifyforCalibrationPeakOrder:
3 (Orange),2(Red),1(Green).ExpandtheParameterssection,then
specifyremainingsettings.

5. PerformaspectralcalibrationusingtheAnyDyedyeset.
6. Createaninstrumentprotocolthatspecifiesthecustomdyeset,thenspecifythe
instrumentprotocolinanassay.

For more
information

72

Refertothespecificationsheetforyourinstrumenttoselectacombinationofcapillary
arrayandpolymerthatprovidetherequiredresolution(seeInstrument
documentationonpage199).

DNA Fragment Analysis by Capillary Electrophoresis

Chapter4 Optimizing Capillary Electrophoresis


3730 Series instruments

3730 Series instruments


Note: Becauseofthecloseproximityofcapillariesonthe96capillary3730xl
instrument,werecommendusingthe48capillary3730instrumentforfragment
analysis.
Note: Sizingprecisionmaybelowerthanexpectedforfragments<50bprunon
3730/3730xlinstrumentswithPOP7polymer.

Run module and


performance

Note: IfyouuseGeneScan1200LIZSizeStandard,downloadanewrunmodule
andoptimizeitbeforeuse.SeeDownloading3730instrumentrunmodulesfromour
websiteonpage48.
Table13 3730 Series instrument run module
3730 Analyzer

Fragment
analysis run
modules
Fragment
Analysis

Resolution

Up to 500 bp resolution with


0.15 bp sizing resolution

Runs/
day
44

3730xl Analyzer

Samples/
day

Genotypes/
day

Samples/
day

Genotypes/
day

4224

84,508

2112

42,254

20 genotypes/sample.

Refertothespecificationsheetforyourinstrumenttoselectacombinationofcapillary
arrayandpolymerthatprovidetherequiredresolution(seeInstrument
documentationonpage199).

Dye sets and


matrix standards

Table18onpage86liststhedyesetsandmatrixstandardseachinstrument.

Creating a custom
dye set

IMPORTANT! WerecommendusingonlyThermoFisherScientificdyes.ThermoFisher
Scientificprovidescalibrationreagentsthathavebeenoptimizedforourdyesets.
NonThermoFisherScientificdyes(ormixedisomersofdyes)havevariableemission
spectraandrequireaspectralcalibrationgeneratedforthespecificdyestocorrectfor
thespectraloverlapbetweenthedyes.Youareresponsibleforobtainingthe
appropriatespectralcalibrationreagentsandforoptimizingcustomdyesetstoensure
thedyelabelsdonotaffectPCRefficiency.
Thesoftwareassumesthefollowingdyecolororder:blue,green,yellow,red,orange.

1. InthenavigationpaneoftheDataCollectionSoftware,click
GA Instruments > ga3730 or ga3130> ProtocolManager.
2. IntheInstrumentProtocolspane,clickNew.TheProtocolEditoropens.
3. IntheProtocolEditor,createaspectralprotocolfortheAny4DyeorAny5Dyedye
set,specifyingtheappropriateprotocolparameters.

4. CreateaSpectralPlateRecordusingthenewlycreatedSpectralInstrument
Protocol.

5. Performaspectralcalibration.

DNA Fragment Analysis by Capillary Electrophoresis

73

Chapter4 Optimizing Capillary Electrophoresis


3730 Series instruments

6. Setthecustomdyesetcalibrationastheactivespectralcalibration:
a. InthetreepaneoftheDataCollectionsoftware,click
GA Instruments > ga3130xl orga3130 > instrument
name >
SpectralViewer.
b. IntheDyeSetdropdownlist,selectthecustomdyeset.
c. IntheListofCalibrationsforDyeSetdropdownlist,selectthespectral
calibrationyouwanttouse.Thespectralprofileandrawdataisdisplayed.

d. ClickSet.
e. Createaninstrumentprotocolthatspecifiesthecustomdyeset.

For more
information

74

Refertothespecificationsheetforyourinstrumenttoselectacombinationofcapillary
arrayandpolymerthatprovidetherequiredresolution(seeInstrument
documentationonpage199).

DNA Fragment Analysis by Capillary Electrophoresis

Chapter4 Optimizing Capillary Electrophoresis


3130 Series instruments

3130 Series instruments


Run modules and
performance

Note: IfyouuseGeneScan1200LIZSizeStandard,downloadanewrunmodule
andoptimizeitbeforeuse.SeeDownloading3130instrumentrunmodulesfromour
websiteonpage48.
Table14 3130 Series instrument run modules and resolution
24-hr throughput

Fragment analysis run


modules

Array
length

Polymer

Fragment Analysis 22_POP4

22 cm

SNP22_POP4
Fragment Analysis 36_POP7

Performance

Run
time

3130
Analyzer
GT

3130xl
Analyzer
GT

Resolution

POP-4

20 min

5,760

23,040

400 bp

0.50

22 cm

POP-4

15 min

3840

15,360

120 bp

0.50

36 cm

POP-7

35 min

3280

13,120

500 bp

0.15

36 cm

POP-4

45 min

2560

10,240

500 bp

0.15

HID Fragment Analysis


36_POP4

36 cm

POP-4

45 min

2560

10,240

500 bp

0.15

SNP36_POP4

36 cm

POP-4

30 min

3840

15,360

120 bp

0.15

50 cm

POP-7

50 min

2240

8,960

500 bp

0.15

50 cm

POP-4

65 min

1760

7,040

500 bp

0.15

50 cm

POP-6

90 min

1280

5,120

500 bp

0.15

Fragment Analysis 36_POP4

Fragment Analysis 50_POP7


Fragment Analysis 50_POP4
Fragment Analysis 50_POP6

SD

20 genotypes/injection.
Standard deviation: 1 base pair (bp) resolution at 99.99% accuracy.
10 genotypes/injection.

Refertothespecificationsheetforyourinstrumenttoselectacombinationofcapillary
arrayandpolymerthatprovidetherequiredresolution(seeInstrument
documentationonpage199).

Dye sets and


matrix standards

Table18onpage86liststhedyesetsandmatrixstandardseachinstrument.

Creating a custom
dye set

IMPORTANT! WerecommendusingonlyThermoFisherScientificdyes.ThermoFisher
Scientificprovidescalibrationreagentsthathavebeenoptimizedforourdyesets.
NonThermoFisherScientificdyes(ormixedisomersofdyes)havevariableemission
spectraandrequireaspectralcalibrationgeneratedforthespecificdyestocorrectfor
thespectraloverlapbetweenthedyes.Youareresponsibleforobtainingthe
appropriatespectralcalibrationreagentsandforoptimizingcustomdyesetstoensure
thedyelabelsdonotaffectPCRefficiency.
Theprocedureforcreatingcustomdyesetsisthesameastheprocedureforthe3730
Seriesinstruments.Creatingacustomdyesetonpage73.

For more
information

Refertothespecificationsheetforyourinstrumenttoselectacombinationofcapillary
arrayandpolymerthatprovidetherequiredresolution(seeInstrument
documentationonpage199).

DNA Fragment Analysis by Capillary Electrophoresis

75

Chapter4 Optimizing Capillary Electrophoresis


310 instruments

310 instruments
Run modules and
performance

Note: IfyouuseGeneScan600LIZSizeStandard,optimizetherunmodulebefore
use.SeeOptimizingthe310instrumentrunmoduleonpage46.
Table15 310 instrument run module

Fragment
analysis run
modules
GS STR POP4

Resolution

Samples/day

Genotypes/day

1 base detection up to 250 bases with 0.15 SD

4-dye

>57

720 genotypes

2 base detection 250 to 350 bases with 0.3 SD

5-dye

>57

960 genotypes

15 genotypes/run.
20 genotypes/run.

Refertothespecificationsheetforyourinstrumenttoselectacombinationofcapillary
arrayandpolymerthatprovidetherequiredresolution(seeInstrument
documentationonpage199).

Dye sets and


matrix standards

Table18onpage86liststhedyesetsandmatrixstandardseachinstrument.

Optimizing sample loading concentration


Note: Sampleoverloadingcanclogcapillaries.
OptimizetheratioofsampletosizestandardandHiDiformamideusingthe
valueslistedbelowasastartingpoint.

Components
Sample
Size standard
Hi-Di

Formamide

3500 Series, 3730 Series, and


3130 Series instruments

310 instrument

0.5 L per reaction

0.5 L per reaction

0.5 L per reaction

0.5 L per reaction

9.0 L per reaction

11.0 L per reaction

Hi-Di Formamide (Part no. 4311320) is purchased separately from the size standard.

Ifyouanticipateanextremelyhighsampleconcentration,rundilutionsofthe
sample.Ifthesignalistoostrong,youcanfurtherdilutethesampleoryoucan
decreasethesampleinjectiontimeand/orinjectionvoltage.
Ifthesignalistooweak,firsttryincreasingthesignalbyincreasingthesample
injectiontimeorvoltage.
Tooptimizethesignalintensityforagivensample,injectthesamesample
multipletimesusingarangeofinjectionparameters.
Ifthesignalintensityisstilltooweakortheresolutionispoor,concentratethe
sample.

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Optimizing signal intensity

Ifthesignalintensityistoolowafterconcentration,seeDesaltingonpage190.
Differentdyesemitdifferentfluorescenceintensities.Therefore,concentrationsof
PCRproductsmayhavetobeincreasedordecreaseddependingonrelative
fluorescenceintensityduringelectrophoresis.(SeeEmissionandabsorption
(excitation)wavelengthsandrelativeintensitiesonpage38).

Optimizing signal intensity


Optimal detection
ranges

ThermoFisherScientificgeneticanalyzerscanconvertalimitedrangeoffluorescence
signalintodigitalvalues.Foroptimalresults,ensurethesignalintensitiesarewithin
therangeslistedbelow.
Table16 Signal intensity ranges and fluorescence saturation

Balancing
size-standard and
sample-peak
intensities

Instrument

Recommended signal
intensity range

Fluorescence saturation

3500 Series

17510,000 RFU

30,000 RFU

3730 Series

15010,000 RFU

30,000 RFU

3130 Series

1504,000 RFU

8000 RFU

310

1504000 RFU

8000 RFU

Theintensityofsizestandardpeaksshouldbe30to100%oftheintensityofsample
peaks.Dilutesamplesbeforepreparingcapillaryelectrophoresisreactionstobalance
thesignalintensities.Intheexamplebelow,theundilutedsampleyieldsthecorrect
sizestandardtopeakintensityratio.

Undiluted

Size-standard
peaks should be
30 to 100% of the
intensity of sample
peaks

Diluted 1:4

Diluted 1:6

DNA Fragment Analysis by Capillary Electrophoresis

77

Chapter4 Optimizing Capillary Electrophoresis


Optimizing electrokinetic injection parameters

If signal intensity is
above the detection
range

Whenthesignalintensityofapeakistoohigh,theinstrumentcannotmeasurethetrue
valueofthesignalandconsequentlycannotcompensateforthespectraloverlap
betweenthedyes.Asaresult,artifactpeakscalledbleedthroughorpulluppeaks
canappearbeneaththesamplepeaks.Thesepulluppeakscancorruptboth
automatedsizing(becauseextrapeaksarepresentinthesizestandarddyecolor)and
theanalysisofsamples(becausethesizestandardispresentineachsample).
Ifsignalintensityishigh,youcan:
DilutethetemplatebeforePCR
Dilutetheamplifiedsamplebeforeaddingtoformamide
Decreasethesampleinjectiontimeand/orinjectionvoltage

If signal intensity is
below the detection
range

Whensignalistoolow,thesignaltonoiseratioisalsolowandmakesitdifficultto
discriminatebetweensamplepeaksandcommonbackgroundfluctuations.
Ifsignalintensityislow,youcan:
Increasethesampleinjectiontimeorinjectionvoltage
IncreasethevolumeoftemplateaddedtothePCRreaction

Minimizing signal
intensity variation

Tominimizesignalintensityvariations,considertheionicstrengthofsamplesand
consumables.TheamountofDNAinjectedisinverselyproportionaltotheionic
strengthofthesolution.Notethefollowing:
Sampleshighinsaltresultinpoorinjections.
PCRreactionsvaryinefficiency,thereforesomereactionsmayresultinhigher
ionicconcentrationpostamplification.
Conductivityofthesolventusedforinjectionaffectsthesampleinjectionandcan
causevariationinpeakheight.
Therecommendedinjectionsolvent,HiDiFormamide,ishighlydeionized
formamide,formulatedwithastabilizer.StorageofHiDiFormamideis
importantinmaintainingthequalityandconductivityofthesolvent.SeeHi
DiFormamidestorageonpage82.
Formoreinformation,seeIrregularsignalintensitytroubleshootingonpage164.

Optimizing electrokinetic injection parameters


Electrokineticinjectionparametersaffectdataquality,runtorunprecisioninsizing,
andreproducibilityintheamountofsampleloaded.Optimizeparameterstoinject
sufficientDNAtoyieldpeaksofadequateheight(thatis,datawithagoodsignalto
noiseratio)whilemaintainingtheresolutionandprecisionrequiredbythe
application.
TheDataCollectionSoftwareincludesrunmoduleswithpresetvaluesforinjection
timesandvoltagesthathavebeenoptimizedforspecificinstrument/polymer/capillary
lengthconfigurations.Thesevaluesareadequateformanyapplications.However,
considermodifyingtheinjectionparametersiftherunmodulesyieldsignalthatistoo
strongortooweakoriftheresolutionispoor.(Themaximumrecommendedinjection
timeis30secondsandthemaximumpossibleinjectionvoltageis15kV.)

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DNA Fragment Analysis by Capillary Electrophoresis

Chapter4 Optimizing Capillary Electrophoresis


Optimizing electrokinetic injection parameters

Definition of
resolution

Theresolution,Rs,oftwopeaksinanelectropherogramisdefinedas:

whereP1andP2arethepeakpositionsmeasuredbelowthepeakapexandW1andW2
arethepeakwidthsmeasuredathalfpeakmaximum.
AnRsvalueof1correspondstofragmentsthatcanbediscriminatedbyonenucleotide.

Optimizing
injection time

Injectiontimeaffectssignalintensityandresolution.
Note: Saltconcentrationcanalsoaffectsignalintensityandresolution.Ifadjusting
injectiontimeandvoltagedoesnotprovideadequatesignalstrength,youmayneedto
concentratethesampleordesaltthesample(Desaltingonpage190).

Attribute

Effect of injection time

Signal
intensity

Signal intensity (as measured both by peak height and by peak area) typically increases linearly with
increasing injection time. However, an n-fold increase in injection time does not result in an n-fold
increase in peak height. In the examples below, no improvement is seen after 10 seconds for the larger
fragment. The signal decreases dramatically after 40 seconds for the smaller fragment. As the injection
time increases, the resolution decreases, leading to increasing peak widths and decreasing peak
heights

Resolution

Increasing the injection time decreases the resolution. As shown below, the negative effect on resolution
is more pronounced for larger fragments.
The decrease in resolution results from an increase in peak width (as opposed to a decrease in peak
separation).

DNA Fragment Analysis by Capillary Electrophoresis

79

Chapter4 Optimizing Capillary Electrophoresis


Optimizing electrophoresis conditions

Optimizing
injection voltage

Injectionvoltageaffectssignalstrength.However,lowervoltages,whichproduce
lowercurrents,areoftenpreferablebecauseinjectiontimingismoreaccurate.Accurate
timingensuresreproducibilityinsampleloading.
Note: Saltconcentrationcanalsoaffectsignalintensityandresolution.Ifadjusting
injectiontimeandvoltagedoesnotprovideadequatesignalstrength,youmayneedto
concentratethesampleordesaltthesample(Desaltingonpage190).

Attribute

Effect of injection voltage

Signal
intensity

Peak height and peak area increase linearly with increasing injection voltage. The figures below show
the effect of increasing the injection voltage from 53 V/cm to 319 V/cm on peak height and peak area,
respectively, for two different-sized fragments.

Resolution

Injection voltage has little effect on peak resolution.

Optimizing electrophoresis conditions


Optimizingelectrophoresisconditions(runtime,runvoltage,runtemperature)can
greatlyimprovedataquality,runtorunprecision,and/orthroughput.Optimize
settingsappropriatefor:
Rangeoffragmentlengths
Requireddegreeofresolution
Typeofgeneticanalysisyouwillbeperforming(forexample,denaturingor
nondenaturingconditions)
Thesettingsintherunmodulesprovidedaresettoensurethefollowing:
Detectionofallfragmentsinthetypicalsizerangefortheapplication
Acceptableruntimes
Acceptableresolution

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Optimizing electrophoresis conditions

Optimizing
run time

Performtrialrunstodeterminetheminimumacceptableruntimeforagivenrun
voltage.Toensurethatyoucollectsufficientdatatoperformanalysis,setthe
electrophoresisruntimeapproximately10%higherthanthemigrationtimeofthe
largestfragmentofinterest.
Thelargestfragmentofinterestisoftenasizestandardpeakthatisneededforsizing
thelargestsamplefragmentsofinterest.Thesetofsizestandardpeaksthat
GeneMapperSoftwareusestogeneratethesizingcurvecanvarywiththesizecalling
method.Ingeneral,besuretoincludethetwosizestandardpeaksimmediately
smallerthanthesmallestfragmentandthetwosizestandardpeaksimmediately
largerthanthelargestsamplefragmentofinterest,ormodifythesizestandard
definitiontoeliminatethepeaksthatarenotpresent.
Note: Forfasterruntimes,youcanalsoincreasetherunvoltage.However,ahigher
runvoltagecandecreasetheresolution.

Optimizing
run voltage

Runvoltagecanaffectmigrationratesandresolutionbecauseitaffectsthespeedat
whichsamplesmigratethroughthecapillary.Iftheymigratetooquickly,thesamples
donotoptimallyseparate.

Attribute

Effect of run voltage

Migration
rates

Higher run voltages yield faster run times but can affect the resolution.

Resolution

In general, resolution is better at lower run voltages.

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Other factors that affect electrophoresis

Optimizing run
temperature

Performnondenaturingapplicationsatlowertemperatures(27to42C).
ProtocolsfordenaturingapplicationsusePOP4orPOP7polymerwithoptimized
runtemperatures.Alteringtheruntemperaturecanaffectmigrationratesand
resolution.

Other factors that affect electrophoresis


Laboratory
temperature and
humidity

Maintainthelaboratorytemperaturebetween15to30C.Aftertheinstrumentissetup
andinoperation,thelaboratorytemperatureshouldnotfluctuatemorethan 2C.The
instrumentcantolerateupto80%noncondensingrelativehumidity.Avoidplacingit
nearheatersorcoolingducts.

Salt concentration,
ionic strength, and
conductivity

SaltanionscompetewithnegativelychargedDNAforentryintothecapillaryduring
electrokineticinjection.Asthesaltconcentrationofasampleincreases,lessDNAwill
enterthecapillary,decreasingthefluorescencesignal.Excesssaltcanalsoprecipitate
theDNAinthesampletubeinthepresenceofformamide.
TheamountofDNAinjectedisinverselyproportionaltotheionicstrengthofthe
solution(Butleret.al.)

Hi-Di Formamide
storage

CAUTION! MixingHiDiFormamidewithwatergeneratesformicacid.
ProperhandlingandstorageofHiDiFormamideiscritical.Forqualityresults:
Aliquotthecontentsfromtheoriginalbottleintoonetimeuse,1.5mLorsmaller
tubes.
Minimizeexposuretoairandfreeze/thawcycles.
IMPORTANT! Donotfreeze/thawmorethantwotimes.Excessivefreeze/thaw
cyclesorstorageat2to8Cformorethan1weekcauseshydrolysisintoformic
acidandformate.Formateionsmigratepreferentiallyintothecapillaryduring
electrokineticinjectioncausingalossofsignalintensity.
EnsurethatyoudonotcontaminateHiDiFormamidewhensettingup
samples.

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Other factors that affect electrophoresis

Storeforupto3monthsat15to25C.
Storeforupto1weekat2to8C.
Thefigurebelowillustratesthevariationinconductanceindifferentquality
formamidesolutions.
Figure18 Effect of formamide quality on conductance

ImproperlystoredHiDiFormamidecancauseavarietyofelectrophoresisproblems.
Forinformation,seeHiDiformamideonpage159.

Polymer handling
and characteristics

IMPORTANT! Donotleavepolymerontheinstrumentmorethansevendays.Polymer
leftontheinstrumentformorethansevendayscausesalossofresolution.Avoid
actionsthatintroducebubblesorparticlesintothepolymer.Dustinthepolymercan
causedataspikes.
Tominimizebubblesandparticlesinpolymer:
Closethepolymercapduringstoragetominimizeexposureofthepolymertoair.
Cleanthepolymerdeliverysystemwithdeionizedwater.
Discardcapillariesthatareexposedtodustoraredriedout.
Changethebufferandwateranddiscardthewastedailyorbeforeeachsetof
runs.
Table17 Polymer characteristics
Instrument

Polymer characteristics

POP-4 Polymer

Less viscous, fast runs

POP-6 Polymer

More viscous, slow runs

POP-7 Polymer
(not for use on 310 instruments)

Less viscous, fast runs

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Understanding spatial calibration

Understanding spatial calibration


AspatialcalibrationmapseachcapillarylocationtotheCCDcamera,whichdetects
thesignalforeachcapillary.

Capillaries

CCD camera positions

Good spatial calibration

Bad spatial calibration

Spatialcalibrationmaximizesdataqualityandaccuracy.
Refertotheinstrumentuserguideforinstructionsonperformingaspatialcalibration.
SeeInstrumentdocumentationonpage199fordocumentpartnumbers.
Performaspatialcalibrationwhenyou:
Installorreplaceacapillaryarray
Temporarilyremovethecapillaryarrayfromthedetectionblock
Movetheinstrument
Openthedetectionblock

Understanding spectral calibration


IMPORTANT! Alwaysvisuallyexaminethedatageneratedbyaspectralcalibration.
Acceptingacalibrationwithpoordatawillyieldinaccurateresultswhenyourun
samples.
Forinformationoncreatingacustomdyesetforspectralcalibration,seethe
instrumentsectionsearlierinthischapter.

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Understanding spectral calibration

Spectral
calibration

Aspectralcalibration(ormatrixon310instruments)allowsthesoftwaretodistinguish
betweendyesbysubtractingoutthespectraloverlapbetweenthedifferentdyes(for
moreinformation,seeMulticomponentanalysiswithfluorescentdyesonpage36).

Before spectral calibration

After spectral calibration

SpectralcalibrationmatrixstandardsareavailablefromThermoFisherScientificin
premixedformforallThermoFisherScientificinstrumentsanddyesets(seeTable18
onpage86).
Thevaluesinamatrixgeneratedbyaspectralcalibrationareuniqueforeach
instrument,foreachdyeset,andforeachspecificsetofrunconditions.TheData
CollectionSoftwareappliesthevaluesinthematrixtothesampledatatoperform
multicomponentanalysis:theseparationofthedyefluorescenceintherawdatafrom
theinstrumenttothedatastoredinthesamplefiles.
Note: For310instruments,youmustmanuallycreateandapplythematrixfileinthe
GeneMapperSoftware.
Refertotheinstrumentuserguideforyourinstrumentforinstructionsonperforming
aspectralcalibrationorcreatinga310matrix.

When to perform
Performaspectralcalibrationrun:
Whenyouuseanewdyesetontheinstrument
Whenyouchangethecapillaryarrayorpolymer
Foreachcombinationofcapillaryarraylengthandeachdyesetthatyouuse
AfterthelaserorCCDcamerahasbeenrealigned/replacedbyaserviceengineer
Ifyouobserveadecreaseinthequalityofraworanalyzeddata(forexample,
pullupand/orpulldownpeakswithadistinctpattern)

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Understanding spectral calibration

Dye set and matrix standards for spectral calibration


Thetablebelowliststheappropriatedyesetandmatrixcalibrationstandard
combinationsforeachinstrument.Forthedyesetsforapplications,seeTable8onpage
41.
Table18 Matrix standards (see page 197 for part numbers)
Matrix
standard

Dye set

3500

3730/
3730xl

3130/
3130xl

310

DS-33

G5

Yes

Yes

Yes

Yes

DS-02

E5

Yes

Yes

Yes

Yes

DS-32

Yes

Yes

Yes

Yes

DS-30

Yes

Yes

Yes

Yes

DS-31

Yes

Yes

Yes

Yes

DS-34

No

No

No

Yes

AnyDye

Custom dye
set you
create

Yes

Yes

Yes

No

Because of the close proximity of capillaries on the 96-capillary 3730xl instrument, we recommend using the
48-capillary 3730 instrument for fragment analysis. For best results, use the G5 dye set with reduced
cross-talk (RCT) configuration.

Evaluating the
calibration results

Usethefollowingcriteriatoevaluatethedata:
QValueorQualityValueMeasurestheconsistencybetweenthefinalmatrix
andthedatafromwhichitwascomputed.AQvalueof1.0indicateshigh
consistencyandthatnopulluporpulldownpeaksweredetected.
ConditionNumberRepresentstheamountofoverlapbetweenthedyepeaksin
theemissionspectraofthedyesinadyeset.AConditionNumberof1.0(lowest
possiblevalue)indicatesthereisnooverlapinadyeset.Theconditionnumber
increaseswithincreasingpeakoverlap.
SpectralprofileShowstheemissionspectraofthedyes.
RawdataTheemissionimageshowsdistinctfluorescenceemissionmaxima,
oneforeachdye(Figure19).
Figure19 Example output from a spectral calibration using a matrix standard

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Understanding spectral calibration

Q Value and
Condition Number
ranges

Troubleshooting
spectral calibration

Matrix standard

Dye set

Quality value

Maximum
Condition Number

User-provided

AnyDye

0.8 (default)

DS-30

0.95

DS-33

G5

0.95

13.5

DS-32

0.95

8.5

DS-31

0.95

DS-02

E5

0.95

Apoororincorrectmatrixresultsintoomuchortoolittlesubtractionofdyespectral
overlapduringdataanalysis.Eachcausesarecognizableelectropherogramanomaly:
Bleedthroughpeaks,alsocalledpulluppeaks(causedbytoolittlesubtraction)
Elevatedinterpeakbaseline(causedbytoomuchsubtraction)
Ifthespectralcalibrationfails,orifthequalityofapassingcalibrationisnot
acceptable,tryoneormoreofthefollowing:
Ensurethatyouusedfresh,properlypreparedandvortexedmatrixstandard.
Old,improperlyprepared,orinsufficientlyvortexedmatrixstandardcancause
lowsignalintensity.
Checkinstrumentstatusforanyrunerrors.
Verifythecorrectrunmodulewasused.Correctasneededandrepeattherun.
Checkthefreshnessandpreparationofreagents.
Checkforpossiblecontaminationofmatrixstandards.
Makesurethattherearenobubblesinthesamplewells.
Verifythatallpeaksweredetected.
Aslowrunningsystemcanpartiallyorcompletelycutoffthebluepeak.Increase
theruntime(instrumentsotherthanthe3500series)orchangereagentsifneeded
andrepeattherun.
Fortroubleshootingthespectralcalibration,refertotheinstrumentuserguideforyour
instrument.

Understanding the
matrix file (310
instruments only)

Purpose of a matrix
Themostintensefluorescenceemittedbyafluorescentlylabeleddyefallswithina
smallwavelengthdetectionrange.However,somefluorescenceemissioninthe
detectionrangesoftheotherdyeswillalwaysoccur.Youcreateamatrixthat
compensatesforoverlapbysubtractingout,inthedetectionrangeofeachdye,the
portionofthesignalduetofluorescencefromotherdyes.
Runeachrelevantdyematrixstandardseparatelytodeterminetheproportional
amountoffluorescencethatisemittedinalldyedetectionregions.Alwayscreatea
newmatrixifrunconditionschange(suchasthepHorpolymertypeand
concentration).

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Understanding spectral calibration

Virtual Filter Set C


VirtualFilterSetC/dyesetDS34isusedon310instruments.Theemissionmaximum
of6FAMdye,therecommendedbluedyefortheVirtualFilterSetC,isverycloseto
thelaserwavelengthof514.5nm.Thus,thewindowforcollectedbluelightintensity
dataisoffsettolongerwavelengthsanddoesnotcontaintheemissionmaximumof6
FAMdye.Theemissionmaximumof6FAMdyeisalsoveryclosetothedetection
regionforthegreenTETdye.MatrixfilesmadeforVirtualFilterSetCareespecially
susceptibletominorchangesinrunconditions.IfyouareusingVirtualFilterSetC,
watchforevidenceofmatrixproblemsandcreateanewmatrixassoonasproblems
appear.

Factors affecting matrix quality


Agingreagents
Buffertypeandconcentration
Polymertype
Denaturingornondenaturingconditions
Runtemperature

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Data Analysis with GeneMapper


Software and Peak Scanner
Software

Overview............................................................ 89

GeneMapperSoftwarefeatures ........................................ 91

PeakScannerSoftwareFeatures ....................................... 92

Workflow ............................................................ 93

GeneMapperSoftwarepeakdetectionsettings........................... 94

GeneMapperSoftwarepeakstartandendsettings....................... 97

HowtheGeneMapperSoftwareperformssizing ......................... 98

GeneMapperSoftwaresizingmethods ................................ 100

Evaluatingdataquality............................................... 104

Overview
Thischaptercontainsgeneralinformationfordataevaluation.Forapplicationspecific
informationondataevaluation,seetheapplicationchapterslaterinthisguide.
GeneMapperSoftwareanalyzesthedatacollectedonThermoFisherScientificgenetic
analyzerstosizeandgenotypeDNAfragments.YoucanalsousetheGeneMapper
Softwaredatatoperformrelativequantitation(formoreinformation,seeChapter9,
RelativeFluorescenceQuantitation(RFQ)onpage 143).
PeakScannerSoftware,canbeusedforpreliminarysizing.

How the software


processes data

BothGeneMapperSoftwareandPeakScannerSoftwareperformanalysison
original.fsafilesgeneratedbytheDataCollectionSoftware.
.fsa files
Baselining
Peak detection
Size matching/Size curve
Genotyping
Quality Value determination

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Overview

Precise versus
accurate sizing

Whenevaluatingsizing,considertwometrics:
Precision(reproducibility)Themeasureoftheabilitytogeneratethesamesize
consistentlyforagivenfragmentobtainedunderthesameconditions.
AccuracyThemeasureoftheabilitytogeneratefragmentsizesthatarecloseto
theactualsizeasdeterminedbysequencing.
ThesizeofaDNAfragmentisalteredbythedyewithwhichitislabeled,andeach
ThermoFisherScientificdyehasadifferentsize.Therefore,afragmentwithaknown
sizemaybesizeddifferentlywhenrunusingThermoFisherScientificdyesand
instruments.Althoughthissizemaynotbeaccuratewhencomparedtotheactual
size,itwillbeprecisewhencomparedtootherfragmentsrununderthesame
conditions.
Notethefollowing:
Sizingdifferencesbetweenvarioustypesofpolymeraremoreapparentfor
sequences<50basepairs(bp).
Smallerfragments(<50bp)runonPOP7polymeron3730/3730xlinstruments
mayhaveslightlylowersizingprecision.

Relative sizing

Thesizeofafragmentiscalculatedbasedonthesizestandardwithwhichit
comigrates.DyelabeledDNAfragmentscanyielddifferentsizeswhenrunwitha
differentinstrument,polymer,capillaryarraylength,orsizestandardasshownbelow.
Highprecisionisimportantinrelativesizing.
Sample 1
310 instrument

Sample 1
3130 instrument

Guidelines for
consistent sizing

Usethesamesizingmethodforallinjections.
Toverify,checktheanalysismethodintheGeneMapperManagerorintheSize
MatchEditorwindow.
Usethesamesizestandardforallsamplesinarun.Youmayneedtomodifythe
sizestandarddefinitionofindividualsamples.
Toverify,overlaythesizestandardpeaksfromallinjectionsordisplaythesizing
curveforeachsamplefile.
VerifythatalldefinedsizestandardpeaksarepresentintheSizeMatchEditor.
Variablerunconditionscanoccasionallycausesizestandardpeaksnottobe
detected,forexampleifarunistoofast,tooslow,orifthesignalintensityofsome
ofthepeaksistoolow.
UseanAnalysisRangethatincludesallthescans(ordatapoints)where
sizestandardpeaksoccurintherawdataofeachsample.
Toverify,checktheanalysismethodintheGeneMapperManager.

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GeneMapper Software features

Autoanalysis and
manual analysis
(GeneMapper
Software only)

TheGeneMapperSoftwareprovidestwoanalysisoptions:
AutoanalysisThesoftwareappliesananalysismethod,sizestandard,and
(optionally)paneltothefragmentanalysisfilesimmediatelyafterDataCollection
Softwarecollectsthedatafromtheinstrument.Theanalysissettingsaresavedin
theGeneMapperSoftwareproject.Youcanreviewtheanalyzeddatausing
GeneMapperSoftware.
ManualAnalysisYouobtainthefragmentanalysisfilesfromthecomputer
connectedtotheinstrument.IftheDataCollectionSoftwareandthe
GeneMapperSoftwareareinstalledonthesamecomputer,youcanimportthe
datafilesintotheGeneMapperSoftware.IftheDataCollectionSoftwareandthe
GeneMapperSoftwareareinstalledondifferentcomputers,moveorcopythe
filestoanothercomputerthathasGeneMapperSoftwareinstalled.Toperform
analysis,youmanuallyapplytheanalysisparameterstothefragmentanalysis
filesintheGeneMapperSoftware.

GeneMapper Software features


Feature

Description

Autoanalysis

Yes, with the corresponding Data Collection Software version

Applications

Amplified fragment length polymorphism (AFLP), loss of heterozygosity (LOH), microsatellite


genotyping, and SNaPshot genotyping.
Analysis of large fragments (~1200 bp), BAC fingerprinting, genetic fingerprinting, multilocus
variant analysis (MLVA), Fragile X assays, biodefense,
T/B-cell clonality assay, bird sex identification, microsatellites, VNTRs, T-RFLP.

Regulatory
compliance

Security and audit features to help users meet 21 CFR Part 11 requirements

Report

Report Manager tools for customized report generation


Customization of the project auto-saving frequency

Analysis

Definition of a linearity range in the analysis methods


Process Quality Values (PQVs) for automated evaluation

Sizing methods

Least Squares, Cubic Spline, Local Southern, and Global Southern

Instrument
software

Supports data generated on 3500 Series, 3730 Series, 3130 Series, and 310 instruments.

Use environment

Multiuser, client-server deployment

GeneMapper Software v4.1 and later includes the ability to record and reapply the Size
Standard Normalization factor calculated in 3500 Series Data Collection Software
Remote auto-analysis and command line operation

Support

Fully supported by Thermo Fisher Scientific

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Peak Scanner Software Features

Peak Scanner Software Features


Overview

PeakScannerSoftwareisanucleicacidsizingsoftwarethatidentifiespeaksand
fragmentsizesforapplicationspecificcapillaryelectrophoresisassays.Thissoftware
allowsyoutoannotatedatawithfunctionssuchaslabeling,merging,andsplitting
peaks.Thesoftwarestoresalleditingandanalysisdataintheoriginal.fsadatafiles
generatedonThermoFisherScientificgeneticanalysisinstruments.
PeakScannerSoftwareisavailablefreeofchargeonwww.lifetechnologies.com.
Note: ThermoFisherScientificdoesnotsupportPeakScannerSoftware.
Usethissoftwarewithdatageneratedon3730Series,3130 Series,and310instruments.
Itisnotcompatiblewithdatageneratedonthe3500Seriesinstrument,whichperforms
fragmentsizingduringdatacollection.

Features

Importandanalyzefragmentanalysissamplefiles(.fsa)fromallcurrently
supportedThermoFisherScientificgeneticanalyzers
Analyzeddata(sizinginformation)iswrittenbacktothesamplefiles(.fsa)
Abilitytoorganizethesamplefilesinaproject
Simultaneousviewingofrawandanalyzeddata
Largefragmentsizingupto1200bp
Abilitytodefinetheexpectedlinearrangeinlargefragmentsizestandardswhere
nonlinearitymightbeexpected
Expandedfeaturesetforeditingpeaksthatincludeslabeling,merging,and
splittingpeaks
Customizablesizingtable
Abilitytooverlaysizingcurvesonanalyzeddata
Abilitytodisplayandprintplotsinthumbnailview
Lightweightsoftwareapplicationwitheasyinstallation
Abilitytoarchiveprojectswithsamplefilesandassociatedreferencedata
(analysismethods,sizestandardsandsoon)fordatasharingpurposes

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Workflow

Workflow
Step
Set up a run file
Define analysis
parameters

GeneMapper Software
1. Create a new project.

Peak Scanner Software

2. Click to add samples to the project.

Import Sample files (drag and drop or add


files function.)

In the Samples tab, specify the analysis


parameters for the samples in the project:

Choose appropriate size standard and


analysis method.

1. In the Analysis Method column, select or


create an analysis method depending on
the application used (AFLP,
Microsatellite, SNaPshot)
2. In the Panel column, select None or
Project Specific Panel.
3. In the Size Standard column, select the
appropriate size standard.
Analyze

Analyze the data by clicking the green arrow


to analyze the samples in the project.

Analyze the data by clicking the green arrow


to analyze the samples in the project.

Review the data

1. (Optional) In the Genotypes tab, review the


Process Quality Value (PQV) columns (BD,
BIN, CC, LPH, OBA, OS, PHR, SHP, SP,
SPA, SPU, and XTLK).

1. View sizes in sizing table and label and/or


edit peaks.

2. Review the size quality and sizing data: In


the Samples tab, examine the Size Quality
(SQ) scores and the size standards.
3. Modify sizing or analysis parameters if
necessary.

2. Save project and print or export results if


necessary.
3. Check sizing quality.
4. (Optional) Modify sizing or analysis
parameters if necessary.

4. Display the samples and genotypes plots.


5. (Optional) Save/print/export the results.

ForalistoftheGeneMapperSoftwaredocumentsavailable,see,Documentation
andSupportonpage 199.

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GeneMapper Software peak detection settings

GeneMapper Software peak detection settings


Peak Amplitude
Thresholds

Onlypeakswithheightsthatexceedthepeakamplitudethresholdvaluesforadye
coloraredetected.

Smoothing

Smoothingoptimizespeaksizeandcanreducethenumberoffalsepeaksdetected:
None(default)appliesnosmoothing.Noneisusefulifthedatadisplaysharp,
narrowpeaksofinterest.
Lightprovidesthebestresultsfortypicaldata.Lightsmoothingslightlyreduces
peakheightintheelectropherogram.Itdoesnotaffecttabulardata.
Heavyisusefulfordatafromslowerrunsthatdisplaybroadpeaksortoavoidthe
detectionofsharpedges.Thisselectionmayreducepeaksizeoreliminatenarrow
peaksintheelectropherogram.Itdoesnotaffecttabulardata.

Baseline Window

TheBaselineWindowadjuststhebaselinesofalldetecteddyecolorstothesamelevel
foranimprovedcomparisonofrelativesignalintensityandhelpstoeliminatenoise
fromthebaseline.
IftheBaselineWindowvalueistoolow,thebaselineapproachesthepeaksand
thedatadisplayshorterpeaks.
IftheBaselineWindowvalueistoohigh,thebaselineistoolowandthedata
displayelevatedandpossiblynotbaselineresolvedpeaks.

Min. Peak Half


Width

TheMin.PeakHalfWidthsettingspecifiesthesmallestfullwidthathalfmaximumfor
peakdetection.
Usealowvalueifthedatadisplaynarrowpeaks.
Ifthevalueishigh,noisespikesareignored.

Polynomial Degree
and Peak Window
Size parameters

UsethePolynomialDegreeandthePeakWindowSizesettingstoadjustthesensitivity
ofthepeakdetection.Youcanadjusttheseparameterstodetectasinglebasepair
differencewhileminimizingthedetectionofshouldereffectsornoise.
Sensitivityincreaseswithlargerpolynomialdegreevaluesandsmallerwindowsize
values.Conversely,sensitivitydecreaseswithsmallerpolynomialdegreevaluesand
largerwindowsizevalues.
Thepeakdetectorcalculatesthefirstderivativeofapolynomialcurvefittedtothedata
withinawindowthatiscenteredoneachdatapointintheanalysisrange.
Usingcurveswithlargerpolynomialdegreevaluesallowsthecurvetomoreclosely
approximatethesignaland,therefore,thepeakdetectorcapturesmorepeakstructure
intheelectropherogram.
Thepeakwindowsizesetsthewidth(indatapoints)ofthewindowtowhichthe
polynomialcurveisfittedtodata.Higherpeakwindowsizevaluessmoothoutthe
polynomialcurve,whichlimitsthestructurebeingdetected.Smallerwindowsize
valuesallowacurvetobetterfittheunderlyingdata.

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GeneMapper Software peak detection settings

Function

Effects of varying
the Polynomial
Degree

Polynomial Degree Value

Window Size Value

Increase sensitivity

Higher

Lower

Decrease sensitivity

Lower

Higher

Thefigurebelowshowspeaksdetectedwithawindowsizeof15datapointsanda
polynomialcurveofdegree2(green),3(red),and4(black).Thediamondsrepresenta
detectedpeakusingtherespectivepolynomialcurves.
Notethatthesmallertrailingpeakisnotdetectedusingadegreeof2(green).Asthe
peakdetectionwindowisappliedtoeachdatapointacrossthedisplayedregion,a
polynomialcurveofdegree2couldnotbefittedtotheunderlyingdatatodetectits
structure.

Polynomial curve of degree 4


(black)
Polynomial curve of degree 3
(red)
Polynomial curve of degree 2
(green)

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Chapter5 Data Analysis with GeneMapper Software and Peak Scanner Software
GeneMapper Software peak detection settings

Effects of
Increasing the
Window Size Value

Inthefigurebelow,bothpolynomialcurveshaveadegreeof3andthewindowsize
valuewasincreasedfrom15(red)to31(black)datapoints.
Asthecubicpolynomialisstretchedtofitthedatainthelargerwindowsize,the
polynomialcurvebecomessmoother.Notethatthestructureofthesmallertrailing
peakisnolongerdetectedasadistinctpeakfromtheadjacentlargerpeaktotheright.

Window size value of 31 (black)


Window size value of 15 (red)

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GeneMapper Software peak start and end settings

GeneMapper Software peak start and end settings


TheSlopeThresholdforPeakStartandSlopeThresholdforPeakEndparameters
adjustthestartandendpointsofapeak.
Thevaluesassignedtotheseparameterscanbeusedtobetterpositionthestartand
endpointsofanasymmetricalpeak,orapoorlyresolvedshoulderingpeaktomore
accuratelyreflectthepeakpositionandarea.
Ingeneral,fromlefttoright,theslopeofapeakincreasesfromthebaselineuptothe
apex.Fromtheapexdowntothebaseline,theslopedecreasesnegativelyuntilit
returnstozeroatthebaseline(seethefigurebelow).
Apex

Baseline

Increasingly
positive slope
(+)
0

Increasingly
negative slope
()
0

Notethefollowing:
Fortypicalorsymmetricalpeaks,useavalueofzero.
Forasymmetricalpeaks,selectvaluesotherthanzerotobetterreflectthe
beginningandendpoints.
Avalueofzerodoesnotaffectthesizingaccuracyorprecisionofanasymmetrical
peak.
Note: Thesizeofadetectedpeakisthecalculatedapexbetweenthestartandend
pointsofapeak.Peaksizedoesnotchangebasedonstartandendsettings.
To move the

Then

Start point of a peak closer


to its apex

Change the Slope Threshold for Peak Start


value from zero to a positive number.

End point of a peak closer to


its apex

Change the Slope Threshold for Peak End


value to a more negative number.

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Example

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Chapter5 Data Analysis with GeneMapper Software and Peak Scanner Software
How the GeneMapper Software performs sizing

How the GeneMapper Software performs sizing


TheGeneMapperSoftwareandthePeakScannerSoftwareusesettingsinthe
analysismethodtosizesamples.
Thissectionprovidesabriefdescriptionofthesizingprocess.Formoreinformation,
seetheGeneMapperSoftwareReferenceandTroubleshootingGuide(Pub.no.4403673).

Size-standard
definitions

Duringsizing,thesoftwarecomparesthesizeofobservedfragmentsforthesize
standardinthesampletotheexpectedfragmentsizeslistedinthesizestandard
definitionusedforanalysis.
TheGeneMapperSoftwareincludesseveralsizestandarddefinitions.Youcanalso
createyourownsizestandarddefinitionfilesordownloadupdatedsizestandard
definitionsfromourwebsite.
Datafrom3500Seriesinstrumentscanbeanalyzedwithasizestandarddefinitionthat
specifiesnormalizationifthedatawascollectedwithanormalizationstandard.

Step 1: Size
matching

Sizematchingusesratiomatching,basedonrelativeheightanddistanceof
neighboringpeaks.Itthenderivesqualityvaluesstatisticallybyexaminingthe
similaritybetweenthetheoretical(fromthesizestandarddefinition)andactual
(observed)fragmentpatterns(seethefigureonthenextpage).
Tocompletethisstepsuccessfully,theanalysissoftwaremustmatchatleastthree
peaks.
Thesoftwareignoresanomalouspeaksthatdonotmatchtheexpectedpatterns.The
softwareconstructsabestfitcurveusingthedatapointsofeachsizestandard
fragmentdetected.Acomparisonbetweenthesizescalculatedfromthebestfitcurve
andthematchedpeaksfromthesizestandarddefinitionusingthearrayofnumbersis
performed.Sizematching(andsubsequentsizing)failsifsignificantdifferencesin
peakpatternsarefound,ifnomatchcanbemadebasedontheexpectedpatterns,orif
allpeaksarenotfound.
Becausethesoftwareusesratiomatching(looksfortheexpectednumberofallelesand
expectedpeakpatternsinsteadofspecificdatapoints),itisnotnecessarytodefinenew
sizestandarddefinitionstoaccommodatemigrationshifts.

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How the GeneMapper Software performs sizing

Determines the expected peak spacing and height ratios


Uses the list of sizes from the Size Standard definition.
Note: The values used are for example only and do not reflect typical size standard values.

Base
pair

50

100
x

200

400

2x

4x

Evaluates peaks in the size standard data


Ignores peaks that do not meet expected pattern (dotted peak).

Base
pair

50

100
x

Data
100
point

200
2x

200

400

Base
pair

50

less than
4x

100
x

400

800

Data
100
point

200
2x

200

400
4x

400

800

Plots the sizing curve


Uses peaks that meet expected pattern

500

Base pair

400
300
200
100
0
0

100 200 300 400 500 600 700 800 900

Data point

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GeneMapper Software sizing methods

Step 2: Sizing curve


and sizing
Factors that affect
sizing

Togeneratethesizecallingcurve,thesoftwareplotstheactualdatapointsofthesize
standardagainsttheexpectedsizeofeachsizestandardpeak.Thesizecallingmethod
determineshowthesizecallingcurveisgeneratedandusedtosizeeachsample.
Thesizingmethod,sizestandarddefinition,orsizestandardusedtogeneratethe
sizingcurve
Welltoreadortimetoreaddifferences
Electrophoresisconditions,suchasruntemperature,voltage,orthedenaturing
abilityoftheseparationmatrix
Polymertype(POP4,POP6,POP7)andconcentration
Capillarylength(22cm,36cm,or50cm)
Instrumentmodelduetodifferencesininstrumentconfiguration

GeneMapper Software sizing methods


Thesizingmethodsavailableinclassicandadvancedmodesintheanalysismethodof
theGeneMapperSoftwareare:
LeastSquares
CubicSplineInterpolation
LocalSouthern
GlobalSouthern
Globalmethods,whichgeneratethebestfitcurvefromallmatchedfragmentsinthe
sizestandard,arelessaffectedthanlocalmethodsbyanomaliesintheruntimesof
singlesizestandardfragments.Doesnotnormalizecapillarytocapillary.
Localmethods,whichgeneratethebestfitcurvefromnearbysizestandarddata
points,arelessaffectedbychangesintheelectrophoresisconditionsorintheanalysis
range.(Achangeintheanalysisrangechangesthesubsetofsizestandardfragments
thatisavailableforgeneratingthesizingcurve.)Normalizescapillarytocapillary.

Least Squares
method

100

Advantages
BothLeastSquaresmethods(2ndOrderand3rdOrder)useregressionanalysisto
buildabestfitsizingcurve.Thiscurvecompensatesforanyfragmentsthatmayrun
anomalously.Asaresult,thismethodnormallyresultsintheleastamountofdeviation
forallthefragments,includingthesizestandardsandthesamples.Dependingon
whetheryouchoosethe2ndor3rdOrderLeastSquaresMethodintheAnalysis
Parametersdialogbox,theresultingsizecurveiseitheraquadraticoracubicfunction.
Thesoftwareusestheknownstandardfragmentsandtheassociateddatapointsto
produceasizingcurvebasedonMultipleLinearRegression.

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Chapter5 Data Analysis with GeneMapper Software and Peak Scanner Software
GeneMapper Software sizing methods

Inthefollowingfigures,youcanseethatinnearlyallinstancesthemobilityofan
individualDNAfragmentiscoincidentwiththebestcurvefitoftheentiredataset.
Stateddifferently,themobilityofmostDNAfragmentsisstrictlylengthdependent.
Thismethodautomaticallycompensatesforfragmentsthatrunanomalously.
GeneMapperSoftwarecalculatesabestfitleastsquarescurveforallsamples,
regardlessofthesizingmethodyouchoose.ThecurveisblackintheStandardSizing
Curvewindow.
Note: AllofthegraphsinthissectionweregeneratedusingGeneScanSoftware
v3.7.1.TheseresultsaresimilartoresultsobtainedwhenyouuseGeneMapper
Softwarev3.5andhigher.

2nd-Order Least Squares sizing curve

Cubic Spline
Interpolation
method

3rd-Order Least Squares sizing curve

TheCubicSplinemethodforcesthesizingcurvethroughalltheknownpointsofthe
selectedsizestandard.Althoughthisenforcementproducesexactresultsforthevalues
ofthestandardsthemselves,itdoesnotcompensateforstandardfragmentsthatmay
runanomalously.
Cubic Spline interpolation sizing curve

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GeneMapper Software sizing methods

Possible local sizing inaccuracy


MobilityofanyDNAfragmentcanbeaffectedbyitssequence,andbysecondaryand
tertiarystructureformation.Ifanyinternalsizestandardfragmenthasanomalous
mobility,theCubicSplinemethodmayexhibitlocalsizinginaccuracy.Forexample:
Assumethatastandardfragmentiscloseinmolecularlengthtoanunknownsample
fragment.Assumefurtherthatthestandardfragmentrunsanomalously.TheCubic
Splinemethodassignstheofficialvaluetothisstandardfragment,eventhoughitmay
beslightlyincorrect.Thesizeoftheunknownfragmentisthenlikelytobecalculated
incorrectlyaswell.
Note: Thismethoddoesnotdeterminetheamountofsizingaccuracyerror.

Local Southern
method

TheLocalSouthernmethoddeterminesthesizesoffragmentsbyusingthereciprocal
relationshipbetweenfragmentlengthandmobility,asdescribedbyE.M.Southern
(1979).
IMPORTANT! FortheLocalSouthernMethodtowork,youmusthaveatleasttwo
sizestandardfragmentssmallerthanyoursmallestunknownfragmentandtwo
sizestandardfragmentslargerthanyourlargestunknownfragment.Ifyoudonot,a
secondorderleastsquarescurveextrapolationwillbeusedtoderivethesizecurve,
insteadofthemethodspecifiedintheanalysismethod.
Local Southern sizing curve

Local Southern Method equation


L=[c/(mm0)]+L0
Theequationattemptstodescribethereciprocalrelationshipbetweenthemobility,m,
andthelength,L0,ofthestandardfragments.

How the Local Southern method works


Thismethod,whichissimilartotheCubicSplinemethod,usesthefourfragments
closestinsizetotheunknownfragmenttodetermineabestfitlinevalue.Onlythe
regionofthesizestandardnearthefragmentofunknownlengthisanalyzed.
Note: Sizeestimatesmaybeinaccurateifanyofthesizestandardfragmentsrun
anomalously.

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GeneMapper Software sizing methods

ThisishowtheLocalSouthernmethodworks:

1. Thefittingconstantsofthecurvearecalculatedforeachgroupofthree
neighboringpointsonthestandard.Aseparatecurveiscreatedforeachsetof
threepoints.

2. Acurveisthencreatedbyusingthreestandardpoints(twopointsbelowandone
pointabovethefragment)andafragmentsizeisdetermined.

3. Anothercurveiscreatedbylookingatanadditionalsetofthreepoints(onepoint
belowandtwopointsabovethefragment)andanothervalueisassigned.

4. Thetwosizevaluesareaveragedtodeterminetheunknownfragmentlength.

Global Southern
method

ThismethodissimilartotheLeastSquaresmethodinthatitcompensatesforstandard
fragmentsthatmayrunanomalously.Themethodcreatesabestfitlinethroughallthe
availablepoints,andthenusesvaluesfoundonthatlinetocalculatethefragment
values.
Global Southern sizing curve

Global Southern method equations


Equation

Description
Attempts to describe the reciprocal
relationship between the mobility, m, and the
length, L0, of the standard fragments.
The fitting constants L0, m0, and c are
calculated by a least-squares fit to minimize
the left side quantity.

How the Global Southern method works


Allpointsinthestandardareweightedequallyandthecurveisnotconstrainedto
passthroughanyspecificpoint.Thesoftwarecananalyzealargerangeoffragment
sizeswiththismethod.
Forbestresults,useasizestandardthathasatleasttwopeakssmallerthanthe
smallestfragmentofinterestandatleasttwopeakslargerthanthelargestfragmentof
interest.

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Chapter5 Data Analysis with GeneMapper Software and Peak Scanner Software
Evaluating data quality

Evaluating data quality


Note: Fordetailedinformationonqualityvaluedetermination,seetheGeneMapper
SoftwareReferenceandTroubleshootingGuidev4.1(Pub.no.4403673).

Examining PQVs

TheGeneMapperSoftwaredisplaysProcessQualityValues(PQVs)intheSamplesor
GenotypestaboftheProjectwindow.
Symbol

Default
Range

Definition
Pass: The sample or genotype passed the PQV test.

0.75 to 1.0

Check: A possible problem exists for the sample or


genotype.

0.25 to 0.75

Low Quality/Fail: There is a strong possibility that a


problem exists for the sample or genotype.

0.0 to 0.25

ReviewtheSQ(SizingQuality)andGQ(GenotypeQuality)resultsforeachsample.
ManyofthePQVscanaffecttheGQresult.
Note: IftheSQPQVis
.

,thesampleisnotsizedorgenotyped,andtheGQPQVis

Werecommendexaminingallsamplesthatproduce
SQflags.

(Check)or

(LowQuality)

ForinformationonconfiguringandinterpretingPQVs,refertotheGeneMapper
SoftwareReferenceandTroubleshootingGuidev4.1(Pub.no.4403673).
ForinformationontroubleshootingSQ
page153.

Criteria for a good


electropherogram

results,seeCheckingdataqualityon

Figure20illustratesanelectropherogramthatmeetsthefollowingcriteria:
Peakheightsare50RFU(peaks<50RFUareconsideredtobenoise).
Idealpeakheightsare:
3500Seriesinstruments:175RFU
3730Series,3130Series,and310instruments:150RFU
Peaksaresharpwithnoshouldersorsplits.
Thepeakscorrespondingtodifferentcolordyesmaynotbeofequalintensity,but
thedataforthelessintensecolorsshouldbeclearlyresolvableathigher
magnification.
Allexpectedpeaksaredetected.
Peaksaresizedproperly(seeSizestandardsonpage42).
Ifyouaregenotyping,samplesareaccuratelygenotyped.
Resultsarereproducible.

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Evaluating data quality

Figure20 Example of a good electropherogram

Ifelectropherogramsdonotmeetthecriteriaabove,seeChapter11,Troubleshooting
onpage 151.

Examining peak
definitions

ToexaminehowGeneMapperSoftwarehasdefinedapeak,select
ViewShowPeakPositions.Thepeakpositions,includingthebeginning,apex,and
endofeachpeak,aretickmarkedintheelectropherogram.

Comparing data

UsethesameGeneScansizestandardlabeledwiththesamedyeforallsamples
inasinglestudy.
Comparepeakareas,heights,andsizesinnucleotidebasesonlyiffragmentsare
labeledwiththesamedye.Formoreinformation,seePreciseversusaccurate
sizingonpage90andRelativesizingonpage90.
Compareonlydatathatiscollectedunderthesameconditions(capillaryarray
length,polymertypeandelectrophoreticrunconditions)forthesamestudy
becausetheseconditionsaffecttherelativesizeofthefragment.

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Evaluating data quality

DNA Fragment Analysis by Capillary Electrophoresis

Microsatellite Analysis

Overviewofmicrosatelliteanalysis ..................................... 107

Applications ........................................................ 110

Instrumentandconsumablerecommendations .......................... 111

Experimentandprimerdesignrecommendations........................ 111

Workflow .......................................................... 112

Dataanalysis ........................................................ 112

Commonproblemswithmicrosatelliteanalysis.......................... 113

Identifyingstutterproductsinmicrosatelliteanalysis ..................... 113

Formoreinformation................................................. 118

Overview of microsatellite analysis


Microsatellitemarkers,alsocalledshorttandemrepeat(STR)markers,are
polymorphicDNAlocithatcontainarepeatednucleotidesequence.Eachrepeatunit
canbe2to7nucleotidesinlength,andallelesdifferbythenumberofrepeats.The
numberofnucleotidesperrepeatunitisthesameforamajorityofrepeatswithina
microsatellitelocus.

Microsatellitemarkersarealsoknownas:
Shorttandemrepeats(STRs)
Simplesequencerepeats(SSRs)
Variablenumbertandemrepeats(VNTRs)

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Chapter6 Microsatellite Analysis


Overview of microsatellite analysis

Principle of the
analysis

Microsatelliteanalysisistheseparationoffluorescentlylabeledfragmentsusing
forwardandreverseprimersanddeterminationoftherelativesizeofthefragments.

APCRprimerpairconsistsoftwooligonucleotides(forwardandreverseprimers),
typically15to30nucleotideslong.Eachprimerhybridizestoitsrespective
complementarystrandoftheDNAtemplatesuchthattheprimerpairflanksthe
regionofinterest.Basedontheapplication,oneorbothoftheprimersmaybelabeled
withafluorescentdye.
Thenumberofrepeatunitsatamicrosatellitelocusmaydiffer,soallelesofmany
differentlengthsarepossibleateachlocus.Themicrosatellitemarkerinthefigure
belowcontainsadinucleotiderepeat.WhenPCRisperformedusingprimersthatflank
theregionofinterest,PCRfragmentsofdifferentsizesaregeneratedbasedonthe
lengthofthedinucleotiderepeat.
Figure21 Different repeats lead to PCR fragments of different length (arrows indicate forward
and reverse primers)

Advantages of
using
microsatellite
markers (loci) in
genetic studies

Severalfeaturesofmicrosatellitesandtheircorrespondingsetofallelesmakethem
idealforuseingeneticstudies:
Theyarepresentinlargenumbers.
Theyarerelativelyevenlyspacedthroughoutthegenomeandoftenphysically
situatednearorwithingenes.
Theyshowavarying,butrelativelyhighmutationraterelativeto
nonmicrosatelliteloci:
MitochondrialDNAevolves5to10timesfasterthansinglecopynuclear
DNA
Microsatellitesevolve100to1000timesfasterthansinglecopynuclearDNA
Themutationrateofmicrosatellitelociis102to106eventsperlocusper
generation(Wan,etal.,2004).Therateisbelievedtobedifferentdependingonthe
numberofnucleotidesintherepeatedunit(EckertandHile,2009).

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Overview of microsatellite analysis

TheirallelesareinheritedinaMendelianmannerandarestableovermultiple
generations.
Theirallelescanbeuniquetospecificpopulations.
Detaileddataonallelicvariation,numberofrepeats,andallelicfrequenciesare
widelyavailableforalargenumberofmicrosatellitemarkers.
Thesmallsizeofmicrosatellitelociimprovesthechanceofobtainingaresult,
particularlyforsamplescontainingverylowamountsofDNAand/ordegraded
DNA.
Thesmallsizerangeofmicrosatellitelocimakesthemidealcandidatesfor
coamplificationwhilekeepingallamplifiedallelessmallerthan350basepairs.
ManymicrosatellitelocicanthereforebetypedfromasinglePCR.
Microsatellitealleleshavediscretesizes,allowingforsimplifiedinterpretationof
results.
PCRbasedtestsarerapid,givingresultsin24hoursorless.
PCRbasedtestsareeasytostandardizeandautomate,ensuringreproducible
results.

Microsatellite
motifs and
distribution

STRstypicallycontain2to7nucleotiderepeats.
VNTRscontain10to100nucleotiderepeatingmotifs.
Figure22 STRs compared to VNTRs

Often,thelengthoftherepeatingunitcorrelateswithitsfrequencywithinagenome.
Forexample,inthehumangenome,mononucleotiderepeatsarethemostcommon
formofmicrosatellitesfound,andpentanucleotideandhexanucleotiderepeatsarethe
leastcommon(Ellegren,2004).
However,thefrequencyofarepeatingunitcanvaryacrossaparticularchromosome
asshowninthefollowingfigure.

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Chapter6 Microsatellite Analysis


Applications

Figure23 SSR density in exonic, intronic and intergenic regions on individual human chromosomes: (a) monomers (b)
dimers; (c) trimers; (d) tetramers; (e) pentamers; (f) hexamers. Blue bars, exons; red bars, introns; yellow bars, intergenic
regions (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC151303/figure/F2/ Copyright 2003, Subramanian et al.; licensee
BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all
media for any purpose, provided this notice is preserved along with the article's original URL.)

Applications
Thelargeselectionofhighlyinformativemarkershasmademicrosatelliteanalysisa
widelyacceptedtoolforthefollowingtypesofstudies:
Linkagemappingstudies
Associationstudies
Populationstudies
Parentageanalysis
Breeding
Custommicrosatelliteassaysareoftenusedfor:
Cancerprogressionanalysis
Phylogeneticstudies
Genomescansforanorganismwherecommercialmarkerpanelsarenotavailable
Populationgeneticsstudies
Paternitytesting
Parentageanalysisforselectivebreeding

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Chapter6 Microsatellite Analysis


Instrument and consumable recommendations

Instrument and consumable recommendations


ThisisaThermoFisherScientificsupportedprotocol.
Thermalcycler:2720,Veriti,orGeneAmp9700
Geneticanalyzer:3500 Series,3130 Series,or310instrument
Polymerandcapillaryarray:seeRunmodulesonpage69forthepolymerand
capillaryarraylengthcombinationssupportedoneachinstrument
600LIZSizeStandard
DS33G5dyeset
AmpliTaqandAmpliTaqGoldDNAPolymerasesaretypicallyusedfor
microsatelliteanalysis.LikeotherDNApolymerases,thesepolymerasesmay
catalyzetheadditionofasinglenucleotide(usuallyA,adenosine)tothe3endsof
thetwostrandsofadoublestrandedDNAfragment.Formoreinformation,see
Additionof3AnucleotidebyTaqpolymeraseonpage33andWitmeretal.,
2003.
IMPORTANT! Throughoutasetofexperiments,usethesameequipment,run
parameters,polymers,dyes,andsoon.Consistentconditionsarerequiredtoavoid
mobilityshiftsthatinterferewithaccurateinterpretationofdata.

Experiment and primer design recommendations


Identifythemarkersforyourstudybyexaminingtheexistingscientificliterature
foraspecificmarkerorcrossspeciesmarker,orbyfollowingamicrosatellite
developmentprotocol(FleischerandLowe,1995;Kandpalet.al.,1994).
Thediscoveryandrandomnamingofnewmicrosatellitemarkersacrossdifferent
organismsatmultipleinstitutionshasledtoinconsistentnomenclaturesfor
microsatellites.Formoreinformationonnomenclaturestandardsforspecific
genomes,gotothenomenclaturewebsiteoftheinstitution,afewofwhichare
listedbelow.
Human:https://iris.ucl.ac.uk
Rat/mouse:http://rgd.mcw.edu/
Fly:http://flybase.org/
Designprimerssotherangeofampliconlengthsacrossmarkersinthestudy
spansiswithinthesizestandardfragmentrange,withtwosizestandardpeaks
precedingthesmallestfragmentofinterestandtwosizestandardpeaks
followingthelargestfragmentofinterest.
Use5endlabeledprimers.Thesuccessofmicrosatelliteanalysisdependsupon
theabilitytodetectsmallmobilitydifferences.Thereproduciblesizingandsharp
peaksobtainedwhenusingthe5endlabelingmethodarecrucialtothesuccess
ofthisapplication.
Ifyouplantomultiplex,designprimerswithsimilarannealing
temperatures ~60C.
Usereverseprimertailingononeprimerineachsetofprimerstohelp
differentiatebetweenpeaksmadebytheforwardandreverseDNAstrandsand
topromoteAaddition.SeeTailonpage 34formoreinformation.
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Workflow

BasedonsampleDNAconcentration,robustnessofthePCR,and/orpeakheights
observedincapillaryelectrophoresis,determinewhetheryouneedtodilutethe
PCRproducts.Dilutionscanrangefromundilutedto1:20inwater.Youcanpool
thedilutedPCRproductsifdesired.
DyelabeledPCRproductsmustbemixedindifferentratiosbecauseeachdyehas
aslightlydifferentfluorescencesignalstrength(seeEmissionandabsorption
(excitation)wavelengthsandrelativeintensitiesonpage38).
Toavoidinaccuraciesassociatedwithpipettingsmallvolumes,prepareamaster
mixofreagents.Preparesufficientmastermixforatleastoneextrareaction
volume.
Storethemastermixinthedarkat2to6Cforupto1month,orat15to25C
forlonger.
Atypicalreactionmayinclude:1LofeachPCRproductand0.5Lofthe
GeneScansizestandardin8.5LofHiDiformamide(fordenaturing
applications)ordistilled,deionizedwater(fornondenaturingapplications).
Mastermixreagentsareoptimizedforcapillaryelectrophoresis,anddiffer
dependingonthecapillaryelectrophoresisinstrumentyouuse.

Workflow
1. Selectprimersandsizestandards:
a. Designandorderprimersforamicrosatelliteapplication.
b. OptimizeamplificationconditionswithmicrosatellitemarkersontestDNA.
c. Orderdyelabeledprimers.

2. PCR
3. Capillaryelectrophoresis
4. Dataanalysis

Data analysis
TheGeneMapperSoftwareincludesaMicrosatelliteDefaultanalysismethodthat
youcanuseasastartingpointforanalysis.
Figure24onpage113showsatypicalmicrosatelliteelectropherogramfromthe
GeneMapperSoftware.
Thenumberofrepeatsforagivenlocusmayvary,resultinginallelesofdiffering
lengths.ThefollowingfigureshowstwodifferentFAMdyelabeledhuman
dinucleotideloci(fromtheGeneMapperSoftwaretutorialdataset)fromtwo
individuals.ThetoppanelillustratesaDNAsamplethatishomozygousatbothloci(a
singlemajorpeakisobservedateachlocus),thebottompanelshowsaDNAsample
thatisheterozygousatthesameloci(twomajorpeaksareobservedateachlocus).

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Common problems with microsatellite analysis

Figure24 Example of microsatellite analysis of two samples by capillary electrophoresis.


Samples have different genotypes as shown by the different peaks for the same marker.

Common problems with microsatellite analysis


Themostcommonlyencounteredproblemsduringmicrosatelliteanalysisare:
Poorornonspecificamplification.SeeOptimizingPCRonpage55andPCR
troubleshootingonpage176.
Incomplete3 Anucleotideaddition.SeeIncomplete3Anucleotideaddition
onpage33.
Stutter.Seethenextsection.
SeeChapter11,Troubleshootingonpage151formoreinformationonstutterpeaks
andplusAproducts.

Identifying stutter products in microsatellite analysis


Overview

DuringthePCRamplificationofdi,tri,andtetranucleotidemicrosatelliteloci,minor
productsthatare1to4repeatunitsshorterthanthemainalleleareproduced.The
minorproductpeaksarereferredtoasstutterpeaks.Stutterpeaksmaybecausedby
polymeraseslippageduringelongation(HaugeandLitt,2003;MurrayandLai,2003).

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Chapter6 Microsatellite Analysis


Identifying stutter products in microsatellite analysis

Stutterpeaksappearasmultiplelowerpeaksthatprecedethetrueallelepeak.These
stutterpeaksdifferinsizefromthetrueallelepeakbymultiplesofthelengthofthe
repeatunit.Thenumberofpeaksandtheirintensitiesareproportionaltothelengthof
therepeatandthenumberofrepeatsinthePCRproduct(Shindeet.al.1993).Shorter
repeatunits(diortri,forex.)generatemorestutter,anddinucleotiderepeatstendto
generatemorestutterpeaksthantrinucleotiderepeats.
Stutterpeakscanalsobecausedbyoffscaledata.Formoreinformation,see
Evaluatingdatawithstutteronpage117.
GeneMapperSoftwareisoptimizedtofilteroutstutterpeaks.

Estimating the
amount of stutter

Youcanestimatethepercentstutterbycalculatingtheratioofthecombinedheightsof
thestutterpeakswiththeheightofthetrueallelepeak.Notethefollowing:
Thelongertherepeatunit,thelessstutterproductproduced.Formicrosatellite
lociwiththesamenumberofrepeatunits,thepercentstutterisgreaterfor
dinucleotidemicrosatellitelocithanitisfortrinucleotidemicrosatelliteloci,and
soon(Walshetal.,1996).
Thefigurebelowillustratesthegreaterstutterindinucleotide(left)ascompared
totetranucleotide(right)repeatloci.Eachlocusishomozygous,withthelargest
peakineachfigurerepresentingthetrueallele.

Thepercentstutterincreaseswithincreasingallelelength(thatis,withincreasing
numberofrepeatunits).However,ifsomeoftherepeatsarepartialrepeats,you
maynotseetheproportionateincreaseinpercentstutter.

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Identifying stutter products in microsatellite analysis

Figure25 Stutter percentages for the FGA and TH01 loci. (Black data points indicate loci labeled
with NED dye.)

Dinucleotide
repeats

Successfulamplificationofdinucleotiderepeatmarkersyieldsallelepeaksand
associatedstutterpeakswithinamaximumrangeofeightbasepairsfromtheallele
peak.Inaddition,thenumberofallelepeaksdependsonwhethertheindividualtested
isaheterozygoteorhomozygote.

Dinucleotide repeats in a homozygous individual


TheGeneMapperSoftwareelectropherogramofadinucleotiderepeatmarkerfroma
homozygousindividual(190bp,190bp)isshowninthefollowingfigure.
Figure26 Stutter peaks in a dinucleotide repeat electropherogram (homozygote)

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Chapter6 Microsatellite Analysis


Identifying stutter products in microsatellite analysis

Thepeaksat188bp,186bp,and184bpshowthetypical2bpstutterpatternseenwith
dinucleotiderepeats.Theyrepresentthe2bp,4bp,and6bpstutterpeaksfromthe
true190bptrueallelepeak.

Dinucleotide repeats in a heterozygous individual (8 bp)


TheGeneMapperSoftwareelectropherogramofadinucleotiderepeatmarkerfroma
heterozygousindividual(139bp,147bp)isshowninthefollowingfigure.Allelesizes
differby8bp.
The2bpstutterpeaktotheleftofeachallelepeakisalwaysoflowerintensitythanthe
allelepeakitself.Thelarger147bpallelepeakisoflowerintensitythanthesmaller
139bpallele.Inheterozygotes,thehighermolecularweightallele(thatis,theallele
peakfurthertotherightinelectropherograms)oftenproducesafluorescencesignalof
lowerintensitythanthelowermolecularweightallele,suggestingalessefficient
amplificationofthelargerfragment.Thisphenomenoncouldalsobecausedby
preferentialinjectionofthesmallerfragments.
Figure27 Stutter peaks in a dinucleotide repeat electropherogram (heterozygote 8 bp)

Dinucleotide repeats in a heterozygous individual (4 bp)


TheGeneMapperSoftwareelectropherogramfromadinucleotiderepeatmarkerofa
heterozygousindividual(185bp,189bp)isshowninthefollowingfigure.Allelesizes
differby4bp.
Figure28 Stutter peaks in a dinucleotide repeat electropherogram (heterozygote 4 bp)

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Identifying stutter products in microsatellite analysis

Whenthedifferencebetweentheallelesizesis4bp,ashiftoccursintheheightratio
betweenthetwoallelepeaks(comparethetwoprecedingfigures).Thefluorescence
signalfromthe4bpstutterofthe189bpalleleisaddedtothesignalfromthe185bp
allele.

Dinucleotide repeats in a heterozygous individual (2 bp)


TheGeneMapperSoftwareelectropherogramfromadinucleotiderepeatmarkerofa
heterozygousindividual(216bp,218bp)isshowninthefollowingfigure.Allelesizes
differby2bp.
Figure29 Stutter peaks in a dinucleotide repeat electropherogram (heterozygote 2bp)

Stutter from
218 bp peak is
added to the
216 bp peak

Stutter band

Whenthedifferencebetweentheallelesizesis2bp,thefluorescencesignalfromthe
2bpstutterofthelargerbasepairalleledoesnotappearasaseparatestutterpeak.It
isaddedtothesignalofthesmallerbasepairallele.

Evaluating data
with stutter

Themultipeakpatternseenwithstutterpeakscancomplicateanalysis,particularlyfor
sampleswithtwoormoreallelesthatarecloseinsize.Forexample,smallpeaksina
positionthatisonerepeatunitsmallerthanthetrueallelecanbeinterpretedeitherasa
stutterpeakorasanalleleinaminorcomponentofamixedsample.Thepossible
presenceofstutterpeaksmakesprecisequantitationespeciallyimportant,toallowthe
GeneMapperSoftwarefilteringalgorithmtointerpretthepeakpatternaccurately.
Thepercentstutterforagivenalleleisreproducibleanddoesnotdependonthe
quantityofinputDNAorthenumberoflociamplifiedduringmultiplexPCR.The
relativereproducibilityofpercentstutterisimportantforafewreasons:
Inmanycases,youcanadjustthePeakAmplitudeThresholdintheanalysis
methodoftheGeneMapperSoftwaretofilteroutstutterpeaksanddetectonly
trueallelepeaks.Foremoreinformation,refertotheGeneMapperSoftware
GettingStartedGuide:MicrosatelliteAnalysis(Pub.no.4403672).
Amplificationswithanabnormallyhighpercentstuttercanindicatemixed
samplesorsomeotherproblemwithPCRamplificationorelectrophoresis.

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117

Chapter6 Microsatellite Analysis


For more information

Is stutter a real
problem?

Stutter,onceunderstood,doesnotposearealproblemformicrosatelliteanalysisand
canaidinallelecallingby:
DistinguishingtrueallelepeaksfromnonspecificPCRproducts.Nonspecific
PCRproductsarenotassociatedwithstutterpeaks.
Identifyingallelesthatfallfaroutsidethereportedallelerange.Thepercent
stutterisoftenspecifictoaparticularlocus.Youcansometimesidentifyalleles
thatfallfaroutsidethepreviouslyreportedrangeonthebasisofpercentstutter.

For more information


SeeMicrosatelliteapplicationsonpage200.

118

DNA Fragment Analysis by Capillary Electrophoresis

Single Nucleotide Polymorphism


(SNP) Genotyping

OverviewofSNPgenotyping .......................................... 119

SNaPshotMultiplexSystem.......................................... 120

Overview of SNP genotyping


Overview

ASingleNucleotidePolymorphism(SNP)markerconsistsofasinglebasepairthat
variesintheknownDNAsequence,therebycreatinguptofourallelesorvariationsof
themarker.
...TCGTTGTAGCGCTTAGA...
...AGCAACATCGCTAATCT...

...TCGTTGTAACGCTTAGA...
...AGCAACATTGCTAATCT...

The G-C and A-T


base pairs are two
possible alleles of
this SNP site

SNPmarkersoccurinthehumangenomeatafrequencyofabout1inevery1000bp,
withatotalnumberofover10millionSNPmarkersdistributedevenlyoverthe
3 billionbpsofthehumangenome.Theyhavebeenshowntoberesponsiblefor
differencesingenetictraits,susceptibilitytodisease,andresponsetodrugtherapies.
SNPmarkersareexcellentgeneticmarkerstoconstructhighresolutiongeneticmaps.
SNPmarkerscanbegenotypedbyavarietyofmethods.ThermoFisherScientific
productssupportthefollowingmethods:
Singlebaseextension
ShiftedTerminationAssay(STA)primerextension

Applications (SNP)

SomeapplicationsofSNPgenotypinginclude:
Studyofmutationsimplicatedinvariouscancers
Geneticdiseaseresearch
MitochondrialDNAinvestigations
Scrapiesusceptibilityinsheep
Lossofheterozygosity
Assessperformanceinfoodanimalproduction,
DifferentiatedrugandnondrugformsofCannabis

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Chapter7 Single Nucleotide Polymorphism (SNP) Genotyping


SNaPshot Multiplex System

SNaPshot Multiplex System


Components

TheSNaPshotMultiplexSysteminvestigatesuptotenSNPmarkerssimultaneously
byusingPCRamplification,thendideoxysinglebaseextensionofanunlabeled
primer,andthencapillaryelectrophoresis.Afterelectrophoresisandfluorescence
detection,theallelesofasinglemarkerappearasdifferentcoloredpeaksatroughly
thesamesizeintheelectropherogramplot.Thesizeofthedifferentallelepeakswill
varyslightlyduetodifferencesinmolecularweightofthedyes.
Figure30 Overview of the SNaPshot kit assay

Componentsofthesystemare:
SNaPshotMultiplexKitIncludesSNaPshotMultiplexReadyReactionMix,
controlprimermix,andcontroltemplate.
SNaPshotPrimerFocusKitDesignedtodeterminetheapproximate
fragmentsizesgeneratedbyvariousprimersbeforeSNPgenotyping(criticalif
twooligonucleotidesproduceoverlappingsignalswhenrunsimultaneously)and
enablesthesettingoftightlociwindowsinGeneMapperSoftware.
GeneScan120LIZSizeStandardFivedyesizestandardthatisdesignedfor
reproduciblesizingofsmallfragmentanalysisdatageneratedwiththe
SNaPshotMultiplexSystems.Itaccuratelysizessamplesrangingfrom20to
120 nucleotides(nt).WhenusedwithGeneMapperSoftware,theGeneScan
120LIZSizeStandardeliminatestheneedformanualgenotyping.
MatrixStandardSetDS02Usedforspectralcalibration.
GeneMapperSoftwareGenotypeanalysisfordatageneratedwithSNaPshot
MultiplexSystems.

120

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Chapter7 Single Nucleotide Polymorphism (SNP) Genotyping


SNaPshot Multiplex System

Additionally,theSNaPshotPrimerFocusKitallowsrapidassessmentofpotential
SNPoligonucleotides.Youcanpreviewallpotentialsinglebaseextensionproducts
andcalculatethemobilityrateforeachallele.Afterassessingthisdata,youcan
determinetheoptimalcombinationofSNPmarkersformultiplexing.Afteryou
determinethemultiplexformat,youcanusethereferencedatacreatedwiththePrimer
FocusKittoestablishmarkersandbinsetsintheGeneMapperSoftware,andreduce
thetimerequiredtodefineandeditbinsmanually.

Principle of the
analysis

Inthesinglebaseextensiontechnique,aunlabeledprimerisdesignedtoannealtothe
sequenceadjacenttotheSNPsite.Aftertheprimeranneals,thesinglebaseextension
occursbytheadditionofthecomplementarydyelabeledddNTP(dyeterminator)to
theannealedprimer.EachofthefourddNTPsisfluorescentlylabeledwithadifferent
colordye(Figure31).
Figure31 Single-base extension with dye-labeled ddNTPs

Unlabeled primer

Dye-labeled
ddNTPs

Template DNA

TheadditionofddNTPsyieldsmarkerfragmentsforthedifferentSNPallelesthatare
allthesamelength,butvarybycolor.
Afterelectrophoresisandfluorescencedetection,theallelesofasinglemarkerappear
asdifferentcoloredpeaksatroughlythesamesizeintheelectropherogramplot.The
sizeofthedifferentallelepeakswillvaryslightlyduetodifferencesinmolecular
weightofthedyes.

Advantages

Usesunlabeleduserdefinedprimersthatarecustomizedforyourtarget
Offersmultiplexingcapability(upto10plex,regardlessoftheirpositionsonthe
chromosomeortheamountofseparationfromneighboringSNPloci)
Sensitiveallelefrequencydetection(5%)
CompatiblewithallThermoFisherScientificgeneticanalyzers
AutomatedanalysisusingspecificGeneMapperSoftwaredataanalysismodule
Additionally,theSNaPshotkitcanbeusedforavarietyofotherapplications:
BACfingerprinting
DNAmethylation

Applications
(SNaPshot)

Lowtomediumthroughputlinkageandassociationstudies
Singlelocusfragmentanalysis

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Chapter7 Single Nucleotide Polymorphism (SNP) Genotyping


SNaPshot Multiplex System

ScreenandconfirmSNPs
Screenforpriongenemutation

Screen and confirm SNPs


TheSNaPshotMultiplexSystemincludesavarietyofSNaPshotMultiplexKitsused
forSNPscreeningandvalidation.Eachkitoffersaonetubesinglebaseextension/
terminationreagenttolabelDNAfragments.

Screen for Prion gene mutations


ThesinglebasepairsensitivityoftheSNaPshotMultiplexSystemenablesyouto
accuratelyscreensamplesforcodondifferencesinpriongenes.Priondiseasesare
causedbyabnormallyfoldedisoformsofhostencodedproteins.UsetheSNaPshot
MultiplexSystemtoscreenforSNPsinthegenesthatcodefortheseproteins.For
instance,polymorphismsatcodons136,154and171ofthePrPgeneinsheepandgoats
canleadtoabnormallyfoldedisoformsoftheproteinproducttoresultinscrapie.

Instrument and
consumable
recommendations

Thermalcycler:Veriti,orGeneAmp 9700(forfastthermalcyclers,usea1C/
secondramprate),2720
Geneticanalyzer:3500 Series,3730 Series,3130Series,or310instruments
Polymerandcapillaryarray:seeRunmodulesonpage69forthepolymerand
capillaryarraylengthcombinationssupportedoneachinstrument
GeneScan120LIZSizeStandard
DS02dyeset
IMPORTANT! Throughoutasetofexperiments,useallthesameequipment,run
parameters,polymers,dyes,andsoon.Consistentconditionsarerequiredto
avoidmobilityshiftsthatinterferewithaccurateinterpretationofdata.

122

DNA Fragment Analysis by Capillary Electrophoresis

Chapter7 Single Nucleotide Polymorphism (SNP) Genotyping

SNaPshot Multiplex System

Experiment and
primer design
recommendations

ThisisaThermoFisherScientificsupportedprotocol.
Minimumprimerlengthis23nt,howeveritisstronglyrecommendedthat
primersshorterthan36ntbetestedbeforemultiplexing.
HPLCpurificationofprimerslongerthan30ntisrecommended.Heterogeneous
primerpopulationswillleadtomajoranalysisissues.
Eachprimershouldhave23ntcomplimentarytothegDNAsequence.
Use5tailstocreatedifferentlengthprimers.
Addpoly(dGACT)togenerateasizedifferenceofatleast4to6nt.
Primerscanbecomplimentarytothe()strandoftheDNAifthe(+)strandis
difficulttoassay.
Alwaysrunanegativecontrol(notemplateDNA)whenevaluatinganewprimer.

Workflow

1. Designprimers.
2. PreparetemplatebyPCRoftarget,thencleanup
3. PrepareSNaPshotreactions
4. PostextensionbyPCR,thencleanup
5. Capillaryelectrophoresis
6. Analyzedata

Data analysis

TheGeneMapperSoftwareincludesaSNaPshotDefaultanalysismethodthatyou
canuseasastartingpointforanalysis.

For more
information

Fordocumentsandpublications,seeSNPapplicationsonpage201.
AnextensivelistofpublicationsdemonstratingtheutilityoftheSNaPshotMultiplex
Systemisavailableatwww.lifetechnologies.com.
Fororderinginformation,seeSNaPshotKitsonpage198.

DNA Fragment Analysis by Capillary Electrophoresis

123

124

Chapter7 Single Nucleotide Polymorphism (SNP) Genotyping


SNaPshot Multiplex System

DNA Fragment Analysis by Capillary Electrophoresis

Fingerprinting

Overview........................................................... 125

Amplifiedfragmentlengthpolymorphism(AFLP)Analysis.............. 126

Terminalrestrictionfragmentlengthpolymorphism(TRFLP) ............. 131

BacterialArtificialChromosome(BAC)fingerprinting .................... 132

Highcoverageexpressionprofiling(HiCEP) ............................. 136

Intersimplesequencerepeat(ISSR)PCR ................................ 137

Overview
DNAfingerprintingisatechniquethatisusedtoidentifypatternsthatoccuringenetic
markers.Thesefingerprintsarespecifictoparticularorganisms.Anumberof
techniquesareavailableforfingerprinting.

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125

Chapter8 Fingerprinting
Amplified fragment length polymorphism (AFLP) Analysis

Amplified fragment length polymorphism (AFLP) Analysis


Amplifiedfragmentlengthpolymorphism(AFLP)isamappingtechniqueusedto
visualizepolymorphismsingenomicDNA.TheAFLPsystemcombinesthe
restrictionfragmentlengthpolymorphism(RFLP)techniqueandpolymerasechain
reaction(PCR)togeneratealargenumberofamplifiedrestrictionfragmentsfrom
prepared,genomicDNA.Whenseparatedbyelectrophoresis,thesamplesyield
uniquebandpatternsthat,whenvisualizedbysouthernblotorfluorescencebased
fragmentanalysis,canbeusedforhighresolutiongenotyping,polymorphism
detection,orcladistics(Savelkouletal.1999).

Principle of the
analysis

TheAFLPprocedureinvolvesdigestinggenomicDNAtoproduceapopulationof
restrictionfragments,ligationofprimingsites,thenamplifiedbyPCR.(Goeletal.
2006.)ItissometimesconsideredavariationofrandomamplifiedpolymorphicDNA
(RAPD).

Figure32 AFLP analysis


Restriction
digestion

Ligation

Preselective
amplification

Selective
amplification

Electrophoresis

Sample 1

Sample 2
Sample 3

AFLPispossiblebecausetheabundantcomplexityineukaryoticgenomicDNA
meansthatitisstatisticallylikelythatenoughrestrictionfragmentswillbeshort
enoughtosuccessfullyproducePCRampliconsthatyieldauniquefingerprint
profile.

Advantages

126

ThepowerofAFLPanalysisderivesfromitsabilitytoquicklygeneratelarge
numbersofmarkerfragmentsforanyorganism,withoutpriorknowledgeofthe
genomicsequence.Inaddition,AFLPanalysisrequiresonlysmallamountsof
startingtemplateandcanbeusedforavarietyofgenomicDNAsamples.

DNA Fragment Analysis by Capillary Electrophoresis

Chapter8 Fingerprinting

Amplified fragment length polymorphism (AFLP) Analysis

Applications

Fingerprints,orAFLPbandpatterns,canbeusedformanypurposes.Forexample,
AFLPanalysisisoftenusedinplantresearchwherefingerprintscanbecomparedto
determinetheplantvarietyortocomparethesimilaritiesbetweendifferentplant
varieties.
ThermoFisherScientificprovideskitsforperformingAFLPonmicrobesandplants,
andreagentsthatareusefulforperformingAFLPonotherorganisms.Some
additionalapplicationsforAFLPanalysisinclude:
Moleculardiversitystudies(Zhaoetal.2006andJohnsonetal.2005.)
Phylogenystudies(Goeletal.2006.)
Breeding(Zhaoet.al.,ibid.)
Backcrossstudies(Johnsonetal.andGoeletal.,ibid.)
Mappingofclonedfragmentsinbacterialandyeastartificialchromosomes(BACs
andYACs)(Serra,etal.2006,Naimuddin,etal.2004)
Identifyingnewspeciesorsubspecies(Johnsonetal.ibid.,andSavelkoul,etal.
1999.)
TheAFLPkitsavailablefromThermoFisherScientificareoptimizedforplantsand
microbes.However,theyareanexcellentstartingpointforcustomAFLPexperiments
onotherorganisms(suchasfish).ContactyourThermoFisherScientificfield
applicationsspecialistformoreinformationonusingAppliedBiosystemsAFLPkits
toconductexperimentsinorganismsotherthanplantsormicrobes.

Instrument and
consumable
recommendations

ThisisaThermoFisherScientificsupportedprotocol.
Thermalcycler:Veriti(standardmodeonly),GeneAmp9700,2720
Geneticanalyzer:3500 Series,3730 Series,3130Series,or310instruments
Polymer:seeRunmodulesonpage69forthepolymerandcapillaryarray
lengthcombinationssupportedoneachinstrument
Capillaryarray:
3500Seriesinstruments:50cm
3730Series,3130Series,and310instruments:36cm
GeneScan500ROXSizeStandard(includedinkits)
DS32MatrixStandard(DyeSetF)
IMPORTANT! Throughoutasetofexperiments,useallthesameequipment,run
parameters,polymers,dyes,andsoon.Consistentconditionsarerequiredtoavoid
mobilityshiftsthatinterferewithaccurateinterpretationofdata.

Experiment and
primer design
recommendations

DNA extraction and purification


BecauseAFLPanalysisrequiresonlyasmallamountofDNA(50to500ng,ideally
10 to100ng),DNApurificationiscritical.
WerecommendthefollowingkitsforextractingDNAforAFLPanalysis:
PlantAnalysis:DNAzolReagentorPureLinkGenomicPlantDNAPurification
Kit
MicrobialAnalysis:PureLinkGenomicDNAMiniKit

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Chapter8 Fingerprinting
Amplified fragment length polymorphism (AFLP) Analysis

Restriction
InAFLPexperimentsongenomesofunknowncontent,determinewhetherornot
yourgenomicDNArestrictsproperlywithEcoRIandMseIenzymes.
Ingeneral,theRegularPlantGenomeKitmodulesshouldproducequalitygenetic
fingerprintswithgenomesof5x108to6109basepairs,andtheSmallPlantGenome
Kitmoduleswithgenomesof5x107to5108basepairs.
EmpiricalguidelinessuggestthatiftheG+Ccontentofthegenomeis>65%,MseIwill
notgiveasignificantnumberoffragments.OptimalresultsareobtainedwithMseI
whentheG+Ccontentis<50%.EcoRIalsotendstoproducemorefragmentsin
G+Cpoorgenomes.IncaseswhereanorganismsG+Ccontentisunknown,the
effectivenessoftherestrictionenzymesmustbedeterminedempirically.

Primers
Fortheselectiveamplificationstep,theprimersthattargettheEcoRI/Abindingsiteare
fluorescentlylabeledatthe5end.TheprimersthattargettheMseI/Cbindingsiteare
unlabeled.
Youmayneedtooptimizetoidentifyprimercombinationsthatgeneratesufficient
uniquemarkerfragmentsforastudy.
Forexample,thePlantMappingKitscontaineightselectiveforwardprimersandeight
reverseprimerslabeledwiththe5FAM,NED,andJOEdyelabeled
fluorophores(DyeSetF).Thepossiblecombinationsofforwardandreverseprimers
provides128possibleprimercombinationsthathavebeentestedacrossseveralcrop
genomes,facilitatingidentificationoftheoptimalpair(s)foragivenorganismwithout
havingtodesign,synthesize,orperformqualitycontroltestsofcustomprimers.
TheAppendixoftheAFLPPlantMappingProtocol(Pub.no.4303146)showsprimer
combinationsthathavebeensuccessfullyusedforavarietyofplantspeciesandthe
AFLPMicrobialFingerprintingProtocol(Pub.no.402977)showsprimercombinations
thathavebeensuccessfullyusedforavarietyofmicrobialorganisms.(Notethatif
yourorganismofinterestdoesnotappearinthelist,youcanstillconductexperiments
bychoosingprimersfromthemostcloselyrelatedspeciesthatisavailable.)
Ingeneral,thestrategywithAFLPanalysisistogenerateinformativefragments,or
enoughfragmentssothatindividualsaredistinguishable.However,toomany
fragmentscomplicatetheanalysis,soyoumustempiricallydeterminetheoptimum
numberoffragmentsneededforadequatediscrimination.Asageneralrule,itisbest
tohavebetween50and200peaksasthefingerprintafteramplification.

Workflow

1. Restrictiondigestion
2. Ligation
3. Preselectiveamplification
4. Selectiveamplification
5. Capillaryelectrophoresis
6. Dataanalysis

128

DNA Fragment Analysis by Capillary Electrophoresis

Chapter8 Fingerprinting
Amplified fragment length polymorphism (AFLP) Analysis

Data analysis

TheGeneMapperSoftwareincludesanAFLPDefaultanalysismethodthatyoucan
useasastartingpointforanalysis.
Thismethodcontainsanalysisparametersforpatternrecognitionoffragmentsacross
samplestogenerateafingerprintforeverysample.Thismethodcanbeusedto
analyzeanytypeofdatafromfragmentlengthpolymorphismassayssuchasAFLPor
TRFLP.Featuresofthesoftwareusefulforanalysisinclude:
Abilitytogenerateapanel(thecollectionofmarkers)fromsamplefilesthathave
beenaddedtoaproject.
SizingQualityandGenotypingQualityvaluesflagpoorqualitysamplesenabling
easyidentificationanddecreasemanualreview.
Automaticgenerationoffinalmarkergenotypesinastandardbinaryformat
where1representsthepresenceofagivenfragmentwhile0representsthe
absenceofthecorrespondingfragment.
Uptofourprofilesareexpectedforeachsamplebecause:
BoththeforwardandreversePCRprimersmaybefluorescentlylabeled
Tworestrictionenzymesareused
GeneratepanelsandbinsetsusingtheAFLPDefaultanalysismethod.Youcanthen
routinelyanalyzedatausingthispanel.
Achangeinthefragmentprofilecanbeindicatedbytheabsenceofapeakaswellasa
reductionintheheightofapeakwhencomparingdifferentsamples.
ThefollowingtwofiguresareexamplesoftypicalandpolymorphicAFLPreactions.

Figure33 Typical electropherogram of an AFLP reaction

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129

Chapter8 Fingerprinting
Amplified fragment length polymorphism (AFLP) Analysis

Figure34 Polymorphic AFLP peaks

Thesepeakpatternsareautomaticallyconvertedtoatableofbinarymarkergenotypes
(Figure35),whichcanbeexportedandanalyzedforsimilarityandgenerationof
dendrogramsusingastatisticalsoftwarepackageorotherdownstreamanalysis
softwareforthistypeofclusteringanalysis.
Figure35 AFLP genotypes in GeneMapper Software

For more
information

130

Fordocumentsandpublications,seeAFLPapplicationsonpage200.
Fororderinginformation,seeOrderingInformationonpage193.

DNA Fragment Analysis by Capillary Electrophoresis

Chapter8 Fingerprinting
Terminal restriction fragment length polymorphism (T-RFLP)

Terminal restriction fragment length polymorphism (T-RFLP)


Overview

Terminalrestrictionfragmentlengthpolymorphism(TRFLP)analysisisamapping
techniqueusedtostudycomplexmicrobialcommunitiesbasedonvariationinthe
16S rRNAgene(OsbornandMooreet.al.).Itiscultureindependent,rapid,sensitive,
andreproducibleanddoesnotrequiregenomicsequenceinformation.

Principle of the
analysis

InTRFLPanalysis,fluorescentlylabeledDNAisdigestedwithrestrictionenzymes
thathave4basepairrecognitionsites.Thisstepgeneratesfluorescentlylabeled
terminalrestrictionfragments.Thefragmentsinthedigestarethenseparatedby
capillaryelectrophoresis.Profilescanthenbecomparedbetweensamples,ormatched
toadatabaseofknownspecies.

Applications

Examinemicrobialcommunitystructureandcommunitydynamicsinresponseto
changesindifferentenvironmentalparametersortostudybacterialpopulations
innaturalhabitats.
Studyofcomplexmicrobialcommunitiesindiverseenvironmentssuchassoil
(DerakshaniandLukowet.al.),marineandactivatedsludgesystems
(EschenhagenandSchuppleretal.)
Characterizeoralbacterialflorainsalivainhealthysubjectsversuspatientswith
periodontitis(SakamotoandTakeuchietal.).
PreliminaryscreeningofmicroorganismsbeforeanalysisusingApplied
BiosystemsMicroSEQMicrobialidentificationkits.

Instrument and
consumable
recommendations

ThisisaThermoFisherScientificdemonstratedprotocol.
Thermalcycler:Veriti,GeneAmp9700,2720
Geneticanalyzer:3500 Series,3730 Series,3130Series,and310instruments
Polymer:seeRunmodulesonpage69forthepolymerandcapillaryarray
lengthcombinationssupportedoneachinstrument
GeneScan600LIZSizeStandard
DS33MatrixStandard(DyeSetG)
IMPORTANT! Throughoutasetofexperiments,useallthesameequipment,run
parameters,polymers,dyes,andsoon.Consistentconditionsarerequiredtoavoid
mobilityshiftsthatinterferewithaccurateinterpretationofdata.

DNA Fragment Analysis by Capillary Electrophoresis

131

Chapter8 Fingerprinting
Bacterial Artificial Chromosome (BAC) fingerprinting

Experiment and
primer design
recommendations

FollowtherecommendationsforAFLPanalysis.SeeExperimentandprimerdesign
recommendationsonpage127.

Workflow

DNA
extraction

PCR with
labeled
primers
Digestion with
restriction
enzymes

Data
analysis

Electrophoresis

1. DNAisolationandpurification.
2. PCRamplificationandrestrictionenzymedigestion.
3. Separationanddetectionofthedigestedproductsviaelectrophoresis.
4. Analysisofdatatogeneratethefragmentprofileforeachsample.
5. Clusteringanalysisbasedontheprofileofsamplesfromstep4.

Data analysis

TRFLPanalysisusesthesamedataanalysistechniqueasAFLP.SeeDataanalysison
page129.

For more
information

Fordocumentsandpublications,seeAFLPapplicationsonpage200.
Fororderinginformation,seeOrderingInformationonpage193.

Bacterial Artificial Chromosome (BAC) fingerprinting


Overview

BACfingerprintingprovidesanefficientandcosteffectivemethodofcharacterizing
largegenomicfragmentlibrariesforgenomesequencing,positionalcloning,and
physicalmappingefforts.RestrictionendonucleasedigestionofBACclonesfollowed
byfluorescentdyelabelingcanbeusedtogenerateaprofileorfingerprint.Overlap
betweenfingerprintsaresubsequentlyusedtoassemblecontiguoussequences
(contigs)intheconstructionofwholegenomephysicalmaps.Physicalmapsare
importantresourcesforgenomesequencingefforts,positionalcloning,comparative
genomics,andtodeterminethesizeandstructureofgenomes.
TheSNaPshotMultiplexKit(Luoetal.)providesaneffective,easy,andcosteffective
solutionforhighthroughputBACfingerprinting.

132

DNA Fragment Analysis by Capillary Electrophoresis

Chapter8 Fingerprinting

Bacterial Artificial Chromosome (BAC) fingerprinting

Principle of the
analysis

InBACfingerprintinganalysisusingtheSNaPshotMultiplexKit,BACclonesare
subjectedtorestrictionendonucleasetogeneratefragmentsofvariouslengthsthatend
inA,C,G,orT.TheSNaPshotchemistrythenlabelsthefragmentswiththe
correspondingbasesbysinglebaseextensiontocreateadistinctDNAfragment
patternorfingerprintforeachclone.Theclonesarethenmappedbasedontheorder
oftheoverlappingpartsoffingerprintswithotherclonesofthesamegenome.
Figure36 SNaPshot restriction fragment labeling
Recessed strand
Label template

SNaPshot
reaction mix

Color = genotype

Electrophoresis

Table19 Example of possible six-base cutters for restriction endonucleases and dyes used in
the SNaPshot Multiplex Kit
Restriction
endonuclease

ddNTP

Fluorescent
dye

Restriction fragment
color

EcoRI

GAATTC

dR6G

Green

BamHI

GGATCC

dR110

Blue
Yellow

XbaI

TCTAGA

dTAMRA

XhoI

CTCGAG

dROX

Red

None

HaeIII

Applications

Restriction
site

GGCC

WithBACfingerprinting,youcancreatewholegenomephysicalmapsthatare
importantresourcesfor:
Genomesequencing
Positionalcloning
Comparativegenomics

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Chapter8 Fingerprinting
Bacterial Artificial Chromosome (BAC) fingerprinting

Instrument and
consumable
recommendations

ThisisaThermoFisherScientificdemonstratedprotocol.
IMPORTANT! BACfingerprintingisbaseduponpatternrecognition;therefore,data
analysisisfocusedonrelativesizeanddistribution.Werecommendusingadedicated
instrumentplatformtominimizelowrandomerrorcausedbysizingimprecision.
Thermalcycler:Veriti,GeneAmp 9700,2720
Geneticanalyzer:3500 Series,3730 Series,3130Series,and310instruments
Polymer:seeRunmodulesonpage69forthepolymerandcapillaryarray
lengthcombinationssupportedoneachinstrument
Capillaryarray:
3500Seriesinstruments:50cm
3730Series,3130Series,and310instruments:36cm
GeneScan120LIZSizeStandard
DS33MatrixStandard(DyeSetG5)
IMPORTANT! Throughoutasetofexperiments,useallthesameequipment,run
parameters,polymers,dyes,andsoon.Consistentconditionsarerequiredtoavoid
mobilityshiftsthatinterferewithaccurateinterpretationofdata.

Experiment and
primer design
recommendations

134

Protocolsmaydifferbasedonthekindofrestrictionendonucleasesandthe
BAC DNApurificationkitsthatareused.
EnzymaticdigestionandSNaPshotreagentlabelingcanbeperformedinone
tubeorinseparatereactions.

DNA Fragment Analysis by Capillary Electrophoresis

Chapter8 Fingerprinting
Bacterial Artificial Chromosome (BAC) fingerprinting

Workflow

1. Selectivebacterialgrowthofsinglecolonies.
2. BACpurificationbyrestrictionendonucleasedigestion.
3. RestrictionendonucleasedigestionoftheBACcloneswithseveraldifferent
enzymes.

4. SNaPshotreagentlabelingoffragments.Thedyelabeledprimersareboundto
theBACfragmentsbasedontheoverhangsleftbytherestrictionenzymes(see
Table19onpage133).

5. Postextensioncleanupoftheclones(notshownindiagram).
6. Capillaryelectrophoresis.
7. Dataanalysis.
8. Contigconstruction.

Data analysis

SeeBACapplicationsonpage200.

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135

Chapter8 Fingerprinting
High coverage expression profiling (HiCEP)

For more
information

Fordocumentsandpublications,seeBACapplicationsonpage200.
Fororderinginformation,seeOrderingInformationonpage193.

High coverage expression profiling (HiCEP)


Overview

Thehighcoverageexpressionprofiling(HiCEP)methodoffragmentanalysiswas
developedtoaddresstheshortcomingsingeneexpressionprofilingandtoprovidea
sensitivemethodfordetectingalargeproportionoftranscriptsinbothknownand
unknowngenes,withalowfalsepositiverate.
AsanAFLPbasedgeneexpressionprofilingmethod,theHiCEPmethoddoesnot
requiresequenceinformationandhasareducedrateoffalsepositiveswithahigh
degreeofdetectionofbothcodingandnoncodingtranscripts.AfterHiCEPanalysis,
fragmentsofinterestcanbepurifiedandclonedfromagarosegelsandsequencedto
identifythetranscripts.Ifwholegenomesequenceinformationfortheorganismunder
studyisknown,thefragmentsofinterestcanbeidentifiedbybioinformaticprediction
usingthesequenceinformationavailablefrompublicdatabasesandtherestriction
enzymerecognitionsitesusedintheHiCEPworkflow.

Principle of the
analysis

HiCEPisanAFLPbasedmethod.TheanalysisinvolvesdigestinggenomicDNAto
produceapopulationofrestrictionfragments.Primingsitesarethenligatedontothe
endsoftherestrictionfragmentssothattheycanbeamplifiedbyPCR(Goeletal.
2006).

Applications

Fingerprinting

Recommendations

ThisisaCustomerdemonstratedprotocol.Forinformation,refertoHiCEP
applicationsonpage200.

Workflow

1. Synthesis
2. Digestion
3. Adaptorligationandpurification
4. Digestion
5. Adaptorligation
6. SelectivePCR
7. PostPCRpreparation
8. Capillaryelectrophoresis
9. Dataanalysis

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Chapter8 Fingerprinting
Inter-simple sequence repeat (ISSR) PCR

Figure37 HiCEP workflow

For more
information

Fordocumentsandpublications,seeHiCEPapplicationsonpage200.
Fororderinginformation,seeOrderingInformationonpage193.

Inter-simple sequence repeat (ISSR) PCR


Overview

Intersimplesequencerepeat(ISSR)PCRisafastandinexpensivegenotyping
techniquewithawiderangeofuses,includingthecharacterizationofgenetic
relatednessamongpopulations.ISSRPCRisagenotypingtechniquebasedon
variationfoundintheregionsbetweenmicrosatellites.
Inadditiontotheuseoflongfragmentsforaccurateanalysis,thistechniqueprovides
additionalbenefitsoveragarosegels.TheincreasedsensitivityofThermoFisher
Scientificgeneticanalyzersovertraditionalanalysismethodsroutinelyallowsthe
detectionofanorderofmagnitudemorepeaks,andthisincreasedresolutionresultsin
betterdiscriminationbetweenindividualsbeingcomparedinthepopulations.
However,theprimersthataredesignedtoannealtothediortrinucleotiderepeatscan
lackspecificityinPCRandareamajorcontributortoalackofreproducibility.Also,the
lackofcomplexityoftheISSRprimerscanleadtononspecificamplification,
particularlyifcoupledtopoorqualitygDNAextractionmethodsandsuboptimalPCR
amplificationconditions.

Principle of the
analysis

ISSRPCRusesasinglefluorescentlylabeledprimertotargettheregionbetween
identicalmicrosatellites(Figure38).AnISSRPCRprimercomprisesthreeparts:
Afluorescenttag
Eightdinucleotiderepeatunits(or6trinucleotiderepeatunits)
Oneormoreanchornucleotidesdesignedwithadualpurpose:totargettheend
ofamicrosatelliteregionandtopreventprimerdimerization.Morethan
100 primershavebeendevelopedforuseinISSRtechniques(UBCPrimerSet9,
2006 catalog).

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Inter-simple sequence repeat (ISSR) PCR

Figure38 Example inter-simple sequence repeat (ISSR) region

BecauseISSRsaredominantmarkers,theamplifiedregionsinanISSRPCRarescored
asdiallelic.Betweenindividualswithinapopulation,changesintheamplified
productscanarisethroughstructuralchangestotheregion(insertionsordeletions)or
thelossofprimerbindingsites.

Advantages

Fasterandrequiresalowerstartupinvestmentthanothergenotyping
methodologiessuchasAFLPandRFLP.
SeveralstudieshavecomparedAFLPandISSRresultsandhavefoundISSR
preferablebecauseofthereducednumberofprotocolstepsrequiredandthe
smalleramountsofDNAconsumed.
Lessexpensiveandlesstimeconsumingthanmicrosatellitebasedgenotyping.
Noneedtocloneandcharacterizemicrosatellites.
Capillaryelectrophoresisdeliverssignificantlyhigherresolutionthantraditional
agarosegelelectrophoresis,thusincreasingtheamountofinformationobtained
fromeachexperiment.

Applications

ISSRhasbeenusedtoinvestigatemanyplantandanimalspeciesinthefollowing
techniques:
Geneticfingerprinting(BlairandPanaudet.al.1999)
Genetagging(AmmirajuandDholakiaet.al.2001)
Detectionofclonalvariation(LeroyandLeon2000)
Cultivaridentification(WangandWuet.al.2009)
Phylogeneticanalysis(GuptaandSouframanienet.al.2008)
Detectionofgenomicinstability(AndersonandBrenneret.al.2001)
Assessmentofhybridization(WolfeandXianget.al.1998)
TheversatilityofthisgenotypingtechniquemakesISSRusefulforresearchers
interestedindiversefieldssuchasconservationbiologyandcancerresearch.

Recommendations

138

ThisisaCustomerdemonstratedprotocol.Forinformation,refertoISSR
applicationsonpage200.

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Chapter8 Fingerprinting
Inter-simple sequence repeat (ISSR) PCR

Experiment and
primer design
considerations

Cetyltrimethylammoniumbromide(CTAB)gDNAisolationdelivershighand
consistentamplification(DoyleandDoyle,1993).
InDNAamplification,primersandPCRmastermixesshouldbetestedfor
robustnessandconsistencywhenamplifyingISSRtargetsinbothspecies.
Subsequentlythermalcyclingconditionscanberefined,withparticularfocuson
primerannealingtemperatureandprimerannealingtime.
Foradditionalinformationonoptimization,refertoISSRGenotypingofEndangered
PlantsUsinganOptimizedWorkflowonpage200.

Workflow

Data analysis

IntheGeneMapperSoftware:
InthePanelManager,createapanelforeachdyecolor(primer)withbins,
centeredatwholebasepairs,onebasepairwidecoveringtheentirerangeof80to
1200bp(Figure39).
IntheGeneMapperManager,modifytheAFLPAnalysisMethod(Figure40).
Thismethoddetectspeaksaboveaminimumpeakheightasanalleleandapplies
abinarylabelofeither1or0forthepresenceofapeakinaparticularbin.

Figure39 Creating multiple ISSR bins and example of multiple bins centered at whole base pairs for the blue marker in an
ISSR Panel

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Inter-simple sequence repeat (ISSR) PCR

Figure40 Example of the Allele tab settings for an ISSR analysis method

Example analysis
ISSRwasusedtocomparetwosamples:AgaveshawiishawiifromRosaritoandAgave
shawiishawiifromBorder.
Thefigurebelowshowsthedistinctpeakpatternsofthesetwoindividuals.
Figure41 Example ISSR data

Agave shawii shawii


from Rosarito

Agave shawii shawii


from Border

AfteranalyzingthedataintheGeneMapperSoftware,genotypeswereexportedand
evaluatedusingaspreadsheetprogramto:
AssesstheconsistencyofgenotypingforfourreplicateISSRPCRreactionsfor
eachprimeranalyzed.
Calculatetheallelessharedbetweenthereplicates.
Onlythosealleleswith100%concordancewerescoredastrueallelesandusedin
subsequentphylogeneticanalyses.
Truealleledataforeachindividualforeachprimerwereconcatenatedintoasinglelist
ofbinarystates.Thebinarydatawerethenanalyzedusingthephylogeneticsoftware
MrBayes(HuelsenbeckandRonquist;RonquistandHuelsenbeck).

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Inter-simple sequence repeat (ISSR) PCR

ThephylogramsgeneratedfromtheMrBayessoftwareisshownbelow.Itindicates
withhighconfidencethatthreedistinctpopulationsofAgaveshawiishawii(alsoknown
asShawsAgave)existed.
Figure42 Phylogram generated using MrBayes software shows three distinct populations of
Agave. Individuals collected from Rosarito, Arroyo Honda, and Border are shown in gold, grey,
and purple, respectively. Highlighted individuals correspond to the data presented in Figure41
on page 140. Nodes in phylogram with posterior probability values above 95% are considered
to be informative in Monte Carlo Markov Chain (MCMC) Bayesian analysis (MrBayes).

For more
information

Fordocumentsandpublications,seeISSRapplicationsonpage200.
Fororderinginformation,seeOrderingInformationonpage193.

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Inter-simple sequence repeat (ISSR) PCR

DNA Fragment Analysis by Capillary Electrophoresis

Relative Fluorescence Quantitation


(RFQ)

Overview........................................................... 143

Experimentandprimerdesignrecommendations........................ 144

LOHworkflow ...................................................... 145

Dataanalysis ........................................................ 145

Formoreinformation................................................. 146

MicrosatelliteInstability(MSI)andReplicationError(RER)................ 146

Overview
Relativefluorescencequantitation(RFQ)isatechniqueusedinavarietyoffragment
analysisapplicationstocomparepeakheightsacrosssamples.
Relativefluorescenceapplicationscomparepeakheightorareabetweentwosamples.
Commontechniquesinclude:
QualitativeFluorescence(QF)PCR
QuantitativeMultiplexPCRofShortFluorescentFragments(QMPSF)
MultiplexLigationdependentProbeAmplification(MLPA)

Principle of the
analysis

ThedataforanRFQexperimentcanbeobtainedwithmicrosatelliteorAFLP
analysis.
Peakheightorpeakareacanbeusedtocomparedifferencesinthesamemarkeracross
multiplesamples.However,youmayseeadifferenceinresultsdependingonwhether
peakheightorpeakareaisused.
IMPORTANT! VariationsinsignalintensityadverselyaffectsresultsinRFQ
experiments.Forinformationonminimizingvariation,seeOptimizingsignal
intensityonpage77.
AsanexamplemicrosatelliteRFQexperiment,thefigurebelowshowsan
electropherogramofamicrosatellitemarkerinDNAfromahealthyandtumor
sample.Thereducedpeakheightinthetumorsampleindicatespotentiallossof
heterozygosity(LOH)inthesample.

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Chapter9 Relative Fluorescence Quantitation (RFQ)


Experiment and primer design recommendations

Figure43 Example electropherogram of healthy and tumor samples

Applications

Screeningforlossofheterozygosity(LOH)usingmicrosatellitesorSingle
NucleotidePolymorphisms(SNPs)
Aneuploidyassays
Detectionoflargechromosomaldeletions
Multiplexligationdependantprobeamplification(MLPA)

Experiment and primer design recommendations


Recommendations

Donotuseinternallylabeled([F]dNTPlabeled)fragmentsinquantitative
experiments.Variationsintheperfragmentnumberoflabelednucleotidesand
theincreasedpeakspreadingwiththismethodmakerelativequantitation
unreliable.
Formoreinformation,seeMicrosatelliteAnalysisonpage107,andAmplified
fragmentlengthpolymorphism(AFLP)Analysisonpage126.

Minimizing signal
intensity variation

Tominimizevariations,considertheionicstrengthofsamplesandconsumables.The
amountofDNAinjectedisinverselyproportionaltotheionicstrengthofthesolution:
Sampleshighinsaltresultinpoorinjections.PCRreactionsvaryinefficiency,
thereforesomereactionsmayresultinhigherionicconcentrationpost
amplification.
Conductivityofthesolventusedforinjectionwillaffectthesampleinjectionand
cancausevariationinpeakheight.

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LOH workflow

Formoreinformation,see:
Optimizingsignalintensityonpage77
Irregularsignalintensitytroubleshootingonpage164
Relativefluorescencequantitationapplicationsonpage200
IMPORTANT! Preferentialamplificationcandecreasetheaccuracyofrelative
quantitationmeasurements.Formoreinformation,seePreferentialamplificationon
page31.

LOH workflow
1. Designtheprimersandselecttheprimerdyeset.
2. PCR:
RuntwoDNAsamplesfromeachindividual,forexample:
Onefromnormaltissue(N)
Onefromtumortissue(T)
Note: Somenormaltissuecontaminatingthetumortissuesampleistypical.
Run3to4independentinjectionsforeachsample(NandT)toobtain
sufficientlyaccuratequantitativeestimatesforsubsequentdataanalysis.
RuncontrolDNA:
AmplifyatleastonecontrolDNAsampleforeveryroundofPCR
amplification.
RunatleastoneinjectionofamplifiedcontrolDNAforeverysetof
microsatellitemarkersused.
RunatleastoneinjectionofamplifiedcontrolDNAwheneveryou
changethecapillaryorelectrophoresisconditions.

3. Capillaryelectrophoresis.
4. Dataanalysis.

Data analysis
Precise peak
detection

Optimizepeakdetectionparameterstoensureprecisepeakdetection.Formore
information,seeDataAnalysiswithGeneMapperSoftwareandPeakScanner
Softwareonpage89.
Ifnoisepeaksaredetected,increasetheMinimumPeakHalfWidthoruseastronger
smoothingoptionwhenanalyzingnoisydata.

Determining
relative quantities

Youcandeterminetherelativequantitiesoftwo5endlabeledfragmentsby
comparingthecorrespondingpeakareasorpeakheightsonaGeneMapperSoftware
orPeakScannerSoftwareelectropherogram.

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For more information

Assessthereproducibilityofpeakheightandareaforeachnewanalysis.Notethe
following:
Useareaforslowmigratingorwidepeaksathighconcentration.
Useheightforsharppeaksatlowconcentration.
Thereisalinearrelationshipbetweenthemigrationtimeandthereproducibility.
Asthemigrationtimeincreases,thepeakwidthandareaincrease.Therefore,fast
migratingpeaksresultinhigherreproducibilityasmeasuredbythepeakarea.
However,improvedreproducibilitycalculatedusingpeakheighthasbeen
observedasthemigrationtimeincreases(ShihabiandHinsdale,1995).

Determining
relative number of
molecules

Todeterminetherelativenumberofmoleculesoftwodifferentsizedfragments,
calculatetheratioofrespectivepeakareasorheights.Makesuretocomparepeakarea
topeakareaorpeakheighttopeakheight:
Iftwofragmentsaresimilarinsize,comparepeakheights,especiallyifthepeaks
overlapslightly.Ifthepeaksarewelldefined,peakareaandpeakheightwillgive
similarresults.Ifthepeaksareirregularlyshapedorhaveshoulders,peakheights
willoftengivebetterresultsthanpeakareas.
Iftwofragmentsdiffergreatlyinsize,comparepeakareasbecauselargepeaks
tendtospreadconsiderablymorethansmallpeaks.

For more information


Fordocumentsandpublications,seeLOHapplicationsonpage200.
Fororderinginformation,seeOrderingInformationonpage193.

Microsatellite Instability (MSI) and Replication Error (RER)


Microsatelliteinstability(MSI)describesthereducedfidelityduringthereplicationof
repetitiveDNAoftenoccurringintumorcells.Itisthoughttobecausedbystrand
slippageduringDNAreplicationduetomutationsinDNAmismatchrepairgenes.
MSIleadstotheappearanceofmultipleallelesatmicrosatelliteloci.Replicationerror
(RER)isusuallydefinedasMSIatmultiplemicrosatellitemarkersorloci.The
appearanceofnumerousextraallelesatlowermolecularweightsinthetumorsample
(Figure44bottompanel)indicatessignificantgenomicinstability.

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Microsatellite Instability (MSI) and Replication Error (RER)

Figure44 Microsatellite instability identified by lower peak signal intensity in the tumor sample

Normal

Tumor

ThetechniquefordetectingRERinvolvescomparingmicrosatelliteallelesafterPCR
amplificationinnormalandtumorsamplesfromthesamehost.Youcalculatearaw
RERscoreusinganalgebraicformulathatquantifiestherelativestrengthofthe
stutterpeaksinthetwosamplesafternormalizingfordifferencesinPCRefficiency.
Whilebothmicrosatelliteinstabilityandlossofheterozygosityareindicativeof
canceroustissue,ifanelectropherogramshowsRERatagivenmarkerlocation,an
LOHcalculationforthatalleleregioniscomplicatedoreveninvalid(Canzianetal.
1996).WedonotrecommendLOHcalculationsinregionsthatshowclearsignsof
RER.

DNA Fragment Analysis by Capillary Electrophoresis

147

148

Chapter9 Relative Fluorescence Quantitation (RFQ)


Microsatellite Instability (MSI) and Replication Error (RER)

DNA Fragment Analysis by Capillary Electrophoresis

10

Additional Applications

DNAmethylation .................................................... 149

DNA methylation
Thestudyofmethylation/epigeneticsisemergingasanimportantcomponentof
cancerresearch.Inatypicalassaytodetectmethylation,bisulfitetreatmentofDNA
deaminatesnonmethylatedcytosineandconvertsittouracilwhilemethylated
cytosineremainsunreactive.ThesubsequentstepofPCRamplificationconvertsuracil
basestothymine.UsetheSNaPshotsystemtoquantitativelydetectthebase
differencesintreatedanduntreatedsamplestolearnthemethylationstatusofthe
samples.
Formoreinformation,seeMethylationapplicationsonpage200.

DNA Fragment Analysis by Capillary Electrophoresis

149

10

150

Chapter10 Additional Applications


DNA methylation

DNA Fragment Analysis by Capillary Electrophoresis

11

Troubleshooting

Troubleshootingworkflow............................................ 152

Refertothefollowingsectionsfortroubleshootingsolutionsandinformationonhow
eachcomponentofthesystemcanaffectdata:

Checkingdataquality ................................................ 153

Runningcontrolstoisolateaproblem................................... 156

Sampleissues ....................................................... 158

Reagentandconsumableissues ........................................ 159

Instrumentandambientconditionissues................................ 160

Refertothefollowingsectionsforsymptomtroubleshootinginformation:

Symptomsyoumayobserve ........................................... 162

Irregularsignalintensitytroubleshooting ............................... 164

Migrationtroubleshooting ............................................ 168

Abnormalpeakmorphologytroubleshooting ............................ 169

Extrapeakstroubleshooting ........................................... 172

PCRtroubleshooting ................................................. 176

Irregularbaselinetroubleshooting...................................... 178

Instrumentationtroubleshooting ....................................... 180

SizingorSizeQuality(SQ)troubleshooting.............................. 182

GeneMapperSoftwaretroubleshooting ................................ 187

Preamplificationgeltroubleshooting ................................... 190

Refertothefollowingsectionsforprocedurestosolveissues:

Desalting ........................................................... 190

Evaluating310GeneticAnalyzermulticomponentmatrixquality .......... 191

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Chapter11 Troubleshooting
Troubleshooting workflow

Troubleshooting workflow
Problemswithdatacanbecausedduringanystepoftheexperiment.

DNA isolation
and purification
(template)

Sample reactions
(assay workflows
and kit protocols)

Sample plating
Instrument run
setup

Instrument run
Data collection

Data analysis
Interpretation

Whentroubleshooting,followthisworkflowtoidentifytheproblem.

1. Makesureyouunderstandthebasicsoftheexperiment:
Thechemistry
Labelingofthesamples
Howthegeneticanalyzercollectsdata
Howthedataanalysissoftwareperformssizingandpeakdetection
Reviewtheexperimentforerrorsinprimerdesign,samplequantitationand
purification,pipettingproblems,softwarepreferencesettingsandothercommon
mistakes.

2. Examinethedata.Evaluatetheproblemasspecificallyaspossible:
Isitaproblemwiththesamplepeaks,thebaseline,orthepeaksofonlyone
color?
Lookforpatterns:Doestheproblemexistinallpartsoftherunordoesit
affectonlyDNAfragmentsofacertainlength?inaspecificcapillary?ina
certainareaoftheplate?multipleruns?
Istheproblemvisibleinrawdata?analyzeddata?logfiles?
Continuetorefinethedescriptionoftheproblemasspecificallyandthoroughly
aspossible.

3. Ingeneral,checkfirstfortheissuesthatcanberesolvedmosteasily.Review:
Dataquality
Analysissettings
Datacollection
Experimentalsetup
Formoretroubleshootinginformation,seeyourinstrumentandsoftwareuserguides
andthedocumentslistedinDocumentationandSupportonpage 199.

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Checking data quality

11

Checking data quality


Sizing Quality (SQ)
PQV description

TheGeneMapperSoftwareSQPQVreflectsthesimilaritybetweenthefragment
patterndefinedbythesizestandarddefinitionandtheactualdistributionof
sizestandardpeaksinthesampledata.ThemetricoftheSizingQualitytestisa
combinationofseveralvalueswhichmeasurethesuccessofthealgorithmsthat:
Identifyandeliminateprimerpeaksbasedonpeakshape
Performsizematching(ratiomatching)
Makeasizecallingcurveusingthesizingmethodspecifiedintheanalysis
method.(formoreinformation,seeGeneMapperSoftwaresizingmethodson
page 100)

Checking samples
with
yellow and
red SQ samples

ReviewthedataofthesizestandardsthatfailtheSQPQVasdescribedbelow.For
moreinformation,seeSizingorSizeQuality(SQ)troubleshootingonpage 182.

1. IntheSamplestaboftheGeneMapperwindow,click

(AnalysisLow
QualitytoTop)tosortthedatasothatthesamplesthatproducederrorsappearat
thetopofthetable.

2. IntheSamplestab,selecttherowsforthesample(s)thatdisplay

(Check)or

(Fail)intheSQcolumn.

3. Click

(AnalysisSizeMatchEditor)toviewthesizinginformationforthe
selectedsample(s).

4. IntheNavigationPaneoftheSizeMatchEditor,selectasamplefiletodisplaythe
sizingdatafortheassociatedsample.

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Chapter11 Troubleshooting
Checking data quality

5. Reviewthedataforthefollowingqualities:
Signalstrength Thesignalstrength(peakheight)ofallpeaksmustexceed
thePeakAmplitudeThresholdsdefinedintheanalysismethodusedto
analyzethedata.
Correctsizecalls/labels Allpeakslistedinthesizestandarddefinition
mustbecorrectlyidentifiedbythesoftware.Thelabelsabovethepeaksmust
beinsequentialorderfromlefttoright,lowtohigh.
Evennessofsignalstrength Allpeaksshouldhaverelativelyuniform
signalstrengths.
Note: TomagnifytheplotoftheSizeMatchestab,dragthemousecursor(
acrossaregionofthexoryaxis.

Examining the raw


data for
red SQ
Examine the
sample info, raw
data, and EPT trace

Datafor
SQsamplesisdisplayedonlyintheRawDatatab(seeExaminethe
sampleinfo,rawdata,andEPTtracebelow).

1. IntheProjectwindow,selectasampleinthenavigationpane.

2. Checkforerrormessages.
Note: Ifthiserror
messageis
displayedatany
timewhenyou
areusingthe
software,check
theInfotabto
determinethe
error.

3. Reviewthesampleinformation.Ensurethatthecorrectanalysissettingsanddata
collectionsettingwereused.

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Checking data quality

11

4. ClicktheRawDatatab.
Note: TheRawDatatabistheonlyplaceinthesoftwareinwhichyoucanview:
Negativebaselines
Rundatafor
SQsamples
Theexamplebelowillustratesgoodqualityrawdataformultiplexed
microsatellitedata.

5. ClicktheEPTDatatab.Reviewthecurrent,voltage,temperature,andpower
throughouttheelectrophoresisrun.Largefluctuationsinthevaluescanresultin
poorqualitydata.
TheexamplebelowillustratesagoodqualityEPTtrace.Thevaluesforthetrace
maydifferdependingontherunmoduleused,buttheshapeofthetraceshould
besimilartotheexamplebelow.

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Chapter11 Troubleshooting
Running controls to isolate a problem

Note: TracecolorsdifferbetweentheDataCollectionSoftwareandtheGeneMapper
Software.
Formoreinformation,seeInstrumentationtroubleshootingonpage 180

Running controls to isolate a problem


Tosimplifytroubleshooting,ThermoFisherScientificrecommendsthatyourun
controlswitheveryrunformulticapillaryinstrumentsoreachsetofrunson310
instruments.
Inadditiontocontrolsincludedineachrun,youcanrunsizestandards,installation
standards,agarosegels,orDNAtemplatecontrolswhenadditionaltroubleshootingis
required.

Size standard

1. Performarunwithonlysizestandard,usingoneofthedefaultrunmodulesthat
areprovidedwiththesoftware:
a. Vortexthesizestandardfor1minute.
b. Addtoeachwellofaplate:
3500 Series, 3730 Series, and 3130
Series instruments

0.5 uL of size standard

0.5 uL of size standard


9.5 uL of fresh

Hi-Di

310 instruments

Formamide

11.5 uL of fresh Hi-Di Formamide

c. Runtheplate.

2. Examinethepeakmorphology:
Ifthepeakmorphologychanges,forexample,peaksbecomebroader,are
tailing,orarebelow50RFU,thentheproblemmaybeintheinstrument,
reagents,orHiDiFormamide.
Ifthepeakprofilesforthesizestandardalonearesharpandverywell
defined,addyourproducttothesamewellsandrerun.
Ifthepeakmorphologythenchanges,forexamplepeaksbecomebroader,
showtailing,arelessthan50RFU,thencontaminationmaybecontributing
totheproblem.
Note: Thesizestandardpeakheightsareaffectedbythepresenceofsample
becausethesampleintroducessaltandcompetesforentryintothecapillary
duringinjection.
Ifthesizequalityfailsinthepresenceofsample,itindicatesaproblemwiththe
PCRproduct,forexample,itmaycontaintoomuchsalt.

Installation
standard

156

InstallationstandardscontainpooledPCRproductsamplifiedfrommicrosatelliteloci
presentinCEPHindividual1347.

DNA Fragment Analysis by Capillary Electrophoresis

Chapter11 Troubleshooting
Running controls to isolate a problem

11

Toruntheinstallationstandard:
Forallplatforms,youcanloadtheinstallationstandardasaregularrunandview
theresultsintheGeneMapperSoftware.Forinstructions,refertothe
appropriateinstrumentuserguide.
Alternatively,fora3500Seriesinstrument,youcanrunaperformancecheck
whichproducesareportquantifyingeachpeak(refertoyourinstrumentuser
guide).
ThermoFisherScientificcurrentlysuppliesthefollowinginstallationstandardsforits
capillaryelectrophoresisinstruments(seeInstallationstandardsonpage 197forpart
numbers):

If you use...

Then use...

Which uses these dyes:

GeneScan 600 LIZ Size


Standard v2.0

DS-33 GeneScan
Installation Standards

6-FAM, VIC, NED, and


PET dyes

GeneScan 500 LIZ Size


Standard

DS-33 GeneScan
Installation Standards

6-FAM, VIC, NED, and


PET dyes

GeneScan 500 ROX

DS-30 GeneScan
Installation Standards

6-FAM, HEX, and NED


dyes

Agarose gel

RunthePCRproductthroughanagarosegeliftheelectropherogramshows
miscellaneousunexpectedpeakswhichmaybeduetounincorporatedproduct.
Resultsfromthegelwillhelptodetermineifyoursampleiscontaminated.

DNA template
control

YoucanuseaDNAtemplatecontrol(forexample,CEPH134702ControlDNAuseful
forhumantargetprimers)asaprocesscontroltoensurethatsamplepreparation,PCR,
andelectrophoresisyieldtheexpectedresults.
Theresultscanhelpyoudeterminewhetherfailedreactionsarecausedbypoor
templatequality,problemswiththecontrol,orproblemswiththeprimers:

1. RunanagarosegeltoseparatethePCRproducts.
2. Runcontrolprimerwithcontroltemplatetoeliminatecontaminatedreagentsasa
possiblecause.

3. Runcontroltemplatewithyourprimerstoeliminateyourprimersasapossible
cause.

4. Runcontrolprimerswithtemplatetoeliminatetemplateasapossiblecause.
Possible cause if the control fails

Action

Incorrect PCR thermal cycling conditions

Choose correct temperature control


parameters (refer to your instrument user
guide).

Pipetting errors

Calibrate pipettes, attach tips firmly, and


check technique.

Combined reagents not spun to bottom of


tube

Place all reagents in bottom of tube. Spin


briefly after combining.

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Chapter11 Troubleshooting
Sample issues

Possible cause if the control fails

Action

Combined reagents left at room temperature


or on ice for extended periods of time

Put tubes in block immediately after


reagents are combined.

Restriction digest incomplete (AFLP


applications)

Repeat the restriction-ligation.


Make sure that the volume of enzyme added
does not cause the amount of glycerol to be
>5%, which can lead to EcoRI* (star) activity.

Sample issues
Sample
concentration

Ifthesampleconcentrationistoolow,thesignaltonoiseratiomaybetoolowto
discriminatebetweensamplepeaksandbackgroundfluctuations.
IfthesampleDNAconcentrationistoohigh,signalintensitymaybeoffscaleor
saturatedandcancause:
Splitpeaks
Raisedbaseline
Pulluppeakswhichcanaffectsizingandaccuracyofgenotypes
Adjustsampleconcentrationtoensuresignalintensityiswithintherecommended
rangeforyourinstrument:
Instrument

Recommended signal
level

Fluorescence saturation

3500 Series

17510,000 RFU

30,000 RFU

3730 Series

15010,000 RFU

30,000 RFU

3130 Series

1504000 RFU

8000 RFU

310

1504000 RFU

8000 RFU

Ifnecessary,dilutePCRproducts(beforeincludingthesizestandardinthereagent
mix)sothatthefinalallelepeakheightfallsintotherecommendedrangeforthe
instrument.

Sample
contamination

Samplecontaminationcanmimicadegradedcapillary.Youcandetermineifthe
capillaryissueiscausedbysamplecontaminationbyrunningasizestandardand
formamideonly(seeRunningcontrolstoisolateaproblemonpage 156).

Salt concentration

SaltanionscompetewithnegativelychargedDNAforentryintothecapillaryduring
electrokineticinjection.Asthesaltconcentrationofasampleincreases,lessDNAwill
enterthecapillary,decreasingthefluorescencesignal.Excesssaltcanalsoprecipitate
theDNAinthesampletubeinthepresenceofformamide.

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Reagent and consumable issues

11

Reagent and consumable issues


IMPORTANT! Foreverychemical,readtheSafetyDataSheets(SDSs)andfollowthe
handlinginstructions.Wearappropriateprotectiveeyewear,clothing,andgloves.
Note: FortheSDSsofchemicalsnotdistributedbyThermoFisherScientific,contact
thechemicalmanufacturer.

Laboratory water

PoorqualitylaboratorywatersystemsandcleaningreagentscanadverselyaffectPCR
efficiency,sampleresolution,andsignalintensity.

PCR reagents

ExpiredPCRreagentscancausedecreasedDNAtemplateconcentration.

Hi-Di formamide

ImproperlystoredHiDiFormamidecancause:
Incompletedenaturationofboththesizestandardandsamplepeaks
AlteredpHoftheloadingsolution
Tailingpeaks
Artifacts
Decreasedsignal
EnsurethatyoudonotcontaminateHiDiFormamidewhensettingupsamples.
Formoreinformation,seeHiDiFormamidestorageonpage 82.

Polymer

Degradedorexpiredpolymer,orpolymerthatisleftatambienttemperaturefor
>7 days,cancause:
Reducedcapillaryarraylife(thenumberofrunsperarray)
Reducedresolutionduetoincreasedconductivity(oftencausedbythehydrolysis
ofureainthepolymer)
Lowcurrent
Artifactpeaksfromdegradedpolymer
Reducedsizingprecision:
Sizingdifferencesbetweenvarioustypesofpolymeraremoreapparentfor
sequences<50bp.
Fragments<50bprunon3730/3730xlinstrumentswithPOP7polymer
mayhaveslightlylowersizingprecision.
Polymerthatisleftatambienttemperatureforextendedperiodsoftimecancause
microbubblesinthepump.
Coldpolymercancausebubbles.
Ensurethatpolymerisatroomtemperature.Allowpolymertoequilibratetoroom
temperatureandpressure.Loosenthelidsealatleast30to60minutesbeforeuse.Do
notleavethelidoffthepolymerbottle,asdustmaycontaminatestock,causingspikes
indata.
Note: Donotshakepolymerorintroducebubbles.

DNA Fragment Analysis by Capillary Electrophoresis

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11

Chapter11 Troubleshooting
Instrument and ambient condition issues

Size standard

Sizingqualityissuescanbecausedbyadegradedorimproperlystoredsizestandard.
AsizestandardcanbedegradedbyusingimproperlystoredHiDiFormamide
(seeHiDiFormamidestorageonpage 82).

Ionic buffer
strength
(not applicable to
3500 Series
instruments)

Conductivitychangesinthebufferaffecttheruncurrentandcancausethefollowing:
Decreasedsampleresolution
Slowerthanexpectedmigrationofsizestandardpeaks
Lowcurrent
Possiblecausesofbufferissues:
Waterimpurities
Highsaltconcentration
Expiredorincorrectlystoredbuffer

Instrument and ambient condition issues


Capillary array

Degradedcapillaryarrayscancause:
Decreasedsampleresolution
Broad,laggingpeaks
Possiblecausesofdegradedcapillaryarrayperformance:
Capillaryarraylifeisexceeded
Capillaryarrayisleftidleordriesout
PoorqualityDNAorwaterordegradedHiDiformamidehasintroduced
contaminantsthatultimatelyaffectthecurrentflowthroughthecapillaries
Waterwashisnotperformedasrecommended,orcontaminatedwaterisusedfor
thewash
Cloggedcapillary
Ifthesamecapillaryalwaysfails,runHiDiFormamideblanks,thenan
installationstandardorsizestandardascontrolsthroughthecapillary(see
Runningcontrolstoisolateaproblemonpage 156).
Note: Samplecontaminationcanmimicadegradedcapillary.Youcandetermine
ifthecapillaryissueiscausedbysamplecontaminationbyrunningcleanDNA
samplesorthesizestandardaloneasacontrol.
Bubblesinthecapillaries
Arcing

Pump: large
bubbles

Largebubblescanaffectallormanyofthesamplesinarun.
Largebubblesinthepumporblockscanaffectthecurrentandcancausethe
following:
Nocurrentwhenvoltageisapplied(theflowofionsisblockedbythebubble)
Arunstopsduringinitializationiftheinstrumentdetectsunstablecurrent
Leakdetectederrormessageastheairbubblecompresseswhentheplunger
movesdowntofillthearray

160

DNA Fragment Analysis by Capillary Electrophoresis

Chapter11 Troubleshooting
Instrument and ambient condition issues

11

Arcing
Earlylossofresolutiononcertaincapillaries
Iflargebubblesarepresent,youcanusuallyseethemintheupperpolymerblock,near
thearrayend.Refertotheinstrumentuserguideforinformationonremoving
bubbles.

Pump: small
bubbles

Smallmicrobubblesusuallyonlyaffectsinglecapillaries.
Microbubblesinthepolymerpathcancause:
Currentfluctuationsornocurrent
Decreasedresolution
Possiblecausesofsmallbubblesinthepump:
Polymeris:
Notatroomtemperature.Allowpolymertoequilibratetoroomtemperature
andpressure.Crackopenlidsealforatleast30to60minutesbeforeuse.Do
notleavethelidoffthepolymerbottle,asdustmaycontaminatestock,
causingspikesindata.
Newlyinstalled
Valves,syringes,orthearrayportarenotscrewedtightlyinplace
Refertotheinstrumentuserguideforinformationonremovingbubbles.

Pump: polymer
leaks

Polymerleakscancause:
Formationofcrystalswhichintroducecontaminantsthatcanaffecttheconditions
ofyourrun.Ifthepumpisnotadequatelypushingpolymerthroughthearray,the
arraycanclogorbecomecontaminated.
Lossofresolution

Autosampler
misalignment

Autosamplermisalignmentcancauseconsistentfailuresinthesamewellsorrowsofa
plate.

Temperature/
humidity

Drasticchangesinroomtemperatureandhumiditycancausedistinctchangesin
migration.

Matrix/spectral
Issues

Incorrectmatrix/calibration,orusingdifferentconditionstocalibratethanyoudoto
runsamplescancausethefollowing:
Raisedbaseline
Negativepeaks
Peaksunderpeaks(especiallyifthehighestpeakisnotoffscale)
Multipledyecolorsbeingdetectedasonedyecolor(hasbeenobservedwhen
running5dyesampleswitha4dyematrix)

DNA Fragment Analysis by Capillary Electrophoresis

161

11

Chapter11 Troubleshooting
Symptoms you may observe

Symptoms you may observe


Irregular signal
intensity

Symptom
No signal or low signal

164

Signal intensity is too high or oversaturated

166

Ski-slope peak pattern

166

Decreased signal

167

Size-standard signal and sample signal are not balanced

168

Migration issues

Symptom

See page

Size-standard peaks are not migrating as expected during


a normal run time

168

Sizing precision is low

168

Abnormal peak
morphology

Symptom

See page

Poor resolution

169

Loss of resolution

169

Broad, lagging peaks

170

Tailing peaks

170

Uneven peak heights in dyes in multiplexed sample

171

Sudden loss of signal in all samples

171

Multiple dye colors are detected as one dye color

171

Extra peaks

Symptom

See page

Extra peaks

172

Pull-up peaks

173

Data spikes

174

Split peaks

175

Many small extraneous peaks appearing next to a highintensity peak

175

PCR

162

See page

Symptom

See page

Poor priming resulting in weak signal

176

Amplified DNA concentration is lower than expected

176

PCR inhibition

176

Contamination with exogenous DNA

176

Poor amplification, nonspecific amplification

177

DNA Fragment Analysis by Capillary Electrophoresis

Chapter11 Troubleshooting
Symptoms you may observe

Symptom

See page

Poor amplification, nonspecific amplification in restrictionligation experiments (AFLP only)

177

Hairpin secondary structures in PCR primers

177

Primer/dimer formation

178

Irregular baseline

Symptom

See page

Constant elevated signal in raw data

178

Baseline waterfall

178

Noisy baseline

179

Adequate signal strength with noisy data

179

Instrumentation

Symptom

See page

Low or fluctuating current

180

Drop-off of current signal

181

Current too high

180

Instrument has stopped running and red light is on. There


are black marks inside the instrument.

181

Symptom

See page

No sample plot is displayed for a sample with the error No


sizing data

183

Size Match Editor does not display peak data

183

Missing size-standard peaks

183

Smaller size-standard peaks are not labeled

184

Larger size-standard peaks are not present in trace

184

Extra peaks in size-standard trace

185

Sizing failures occur in a regular pattern (the same wells


fail repeatedly)

186

Noise peaks are detected as size-standard peaks

186

Size call inaccurate for known DNA sample

186

Sizing or size
quality

GeneMapper
Software

Symptom

See page

GeneMapper Software error message

187

Error Message: The bin set in the analysis method does not
match the panel used for analysis.

187

al? label or alleles are not falling within bins

188

Allele not labeled

188

DNA Fragment Analysis by Capillary Electrophoresis

11

163

11

Chapter11 Troubleshooting
Irregular signal intensity troubleshooting

Symptom

See page

Data not sorted by name

188

When adding samples to a project, the expected data files


are not listed in the Add Samples to Project dialog box

188

Genotypes tab is grayed

189

Two peaks do not separate and are detected as one peak

189

Irregular signal intensity troubleshooting


Symptom

Possible cause

Action

No signal or low signal

Peak Amplitude Threshold

164

Poor or non-specific
amplification.

See PCR troubleshooting on page 176.

PCR inhibition.

See PCR troubleshooting on page 176.

Sample was prepared with


water instead of Hi-Di
Formamide.

Prepare the sample with Hi-Di Formamide.

Degraded or incorrectly stored


Hi-Di Formamide.

Use fresh, properly stored Hi-Di Formamide. See


Hi-Di Formamide storage on page 82.

Air bubble at bottom of sample


tube.

Centrifuge the plate before running.

See Non-specific amplification on page 32 for


more information.

DNA Fragment Analysis by Capillary Electrophoresis

Chapter11 Troubleshooting
Irregular signal intensity troubleshooting

Symptom
No signal or low signal
(continued)

Possible cause
High salt concentration.

11

Action
Salt preferentially injects smaller fragments and
inhibits injection of larger fragments, so the
majority of salt may have been injected in the first
injection.
Re-inject the sample.
If signal intensity does not increase, see
Desalting on page 190.

Injection time too low.

See Optimizing electrokinetic injection


parameters on page 78.

Sample concentration too low.

See Optimizing sample loading concentration on


page 76.

Sample volume too low.

Sample volume must be 10 L for 3500 Series,


3730 Series, and 3130 Series instruments.
Sample volume must be 12 L for 310
instruments.

Autosampler is misaligned.

3500 Series, 3730 Series, and 3130 Series


instruments: Fill wells with 0.5 L size standard
and 9.5 L sample, then re-inject. If the signal
is still missing, contact Thermo Fisher Scientific
310 instruments: Recalibrate autosampler.

DNA Fragment Analysis by Capillary Electrophoresis

165

11

Chapter11 Troubleshooting
Irregular signal intensity troubleshooting

Symptom
Signal intensity is too high or
saturated

Possible cause

Action

Raw data: pull-down peaks

Zoomed view of figure above

Sample concentration too high.

Decrease sample concentration.


Decrease injection time.

Ski slope peak pattern of


sample peaks but size standard
peak heights do not decrease

Sample concentration too high


in amplification step or
insufficient primer is present.

166

Optimize ratio of DNA template and primer.

DNA Fragment Analysis by Capillary Electrophoresis

Chapter11 Troubleshooting
Irregular signal intensity troubleshooting

Symptom
Decreased signal

11

Possible cause

Action

Degraded or improperly stored


Hi-Di Formamide.

Use fresh, properly stored Hi-Di Formamide. See


Hi-Di Formamide storage on page 82.

Expired or incorrectly stored


reagents.

Use fresh reagents.

Degraded primers.

Store unused primers at 15 to 25C. Do not


expose fluorescent dye-labeled primers to light for
long periods of time.

Size-standard concentration is
too high.

Although the data is still sized properly, decrease


size-standard concentration to balance peaks in
future runs (see Size-standard peak intensity on
page 42).

Size-standard signal and


sample signal are not balanced

DNA Fragment Analysis by Capillary Electrophoresis

167

11

Chapter11 Troubleshooting
Migration troubleshooting

Migration troubleshooting
Symptom
Size-standard peaks are not
migrating as expected
during a normal run time

Possible Cause
Poor quality sample.
Degraded or frozen
polymer.

Action
Prepare fresh buffer (not applicable to 3500 Series
instruments).

Water used to dilute


buffe.r
Poor-quality formamide.
Fluctuations in ambient
temperature and/or
humidity.
Incorrect oven
temperature.
Old array or capillary.
Contaminants.
Low ionic buffer strength.

Sizing precision is low

Incorrect capillary length


(Length to Detector) or run
module was selected.

Specify correct capillary length or run module.

Variation in ambient
temperature causes faster
or slower migration rates.

Ensure ambient temperature is stable.

Analyzing small fragments


<50 bp.

Sizing differences between various types of polymer


are more apparent for sequences <50 bp.
Fragments <50 bp run on 3730/3730xl Series
instruments with POP- 7 polymer may have slightly
lower sizing precision than expected.

168

DNA Fragment Analysis by Capillary Electrophoresis

Chapter11 Troubleshooting
Abnormal peak morphology troubleshooting

11

Abnormal peak morphology troubleshooting


Symptom
Poor resolution

Possible cause

Action

High sample concentration.

Dilute the sample before adding to formamide/


size-standard mix.

Injection time and/or voltage too


high.

See Optimizing electrokinetic injection


parameters on page 78.

Wrong capillary array and/or


polymer used.

Use appropriate capillary array or polymer for your


application.

Incomplete strand separation


due to insufficient heat
denaturation.

Make sure the samples are heated at 95C for


3 to 5 minutes, then immediately placed on ice for
2 to 3 minutes before loading.

Pump, polymer block, or septa


contaminated with chemicals
during cleaning.

1. Perform a water wash. Refer to the instrument


user guide for information.

Incomplete replacement of
polymer between runs.

1. Check the polymer delivery system for leaks,


looking for residue in and around the polymer
block area.

Loss of resolution

2. Replace polymer, buffer, septa and water/waste


with fresh materials.

2. Check the pin valve for signs of arcing on the tip.


Black markings within the block channel are
also a sign of an arcing event.
3. Check for polymer in the anode buffer jar. If you
see evidence of a leak, retighten connections,
then run the sample again.
Sample or reagent is
contaminated.

DNA Fragment Analysis by Capillary Electrophoresis

Use fresh samples and reagents.

169

11

Chapter11 Troubleshooting
Abnormal peak morphology troubleshooting

Symptom
Loss of resolution (continued)

Possible cause
High salt concentration.

Action
Salt preferentially injects smaller fragments and
inhibits injection of larger fragments, so the
majority of salt may have been injected in the first
injection.
Re-inject the sample.
If signal intensity does not increase, see
Desalting on page 190.

Bubbles or debris in polymer


path.

Remove bubbles or clean the polymer path. Refer


to the instrument user guide for information.

Capillary array degrading.

1. Perform a water wash through the polymer


delivery system. Refer to the instrument user
guide for information.
2. Replace the capillary/array.
3. Run a size standard.
4. If the problem is present in the size standard,
replace reagents, then run your samples again.

Samples are degraded because


they have been sitting in the
instrument >24 hours.

Run samples as soon as possible after


preparation.

Expired or degraded polymer,


Hi-Di Formamide, buffer, or
water.

Replace the reagent, then run your samples again.


Use fresh, properly stored Hi-Di Formamide. See
Hi-Di Formamide storage on page 82.

Instrument current problem.

See Instrumentation troubleshooting on


page 180.

Use of non-Thermo Fisher


Scientific reagents.

1. Perform a water wash on all components of the


system using the wizard in Data Collection
Software.
2. Replace reagents with Thermo Fisher Scientific
products.

Broad, lagging peaks

Old or clogged capillary array.

Replace the capillary array or flush the capillary


array with polymer.

Tailing peaks

Degraded or improperly stored


Hi-Di Formamide.

Use fresh, properly stored Hi-Di Formamide. See


Hi-Di Formamide storage on page 82.

170

DNA Fragment Analysis by Capillary Electrophoresis

Chapter11 Troubleshooting
Abnormal peak morphology troubleshooting

Symptom

Possible cause

11

Action

Uneven peak heights in dyes in


multiplexed sample

Sample preparation issues.

Optimize sample preparation and PCR.

Preferential amplification of
PCR products.

see PCR troubleshooting on page 176.

Selection of dyes is not optimal


(for example, a low-intensity
sample peak is labeled with a
low-intensity dye).

Select appropriate dye. For information, see


Points to consider when selecting dyes for custom
primers on page 39.

Concentration of some samples


is too high.

Adjust the pooling ratio before PCR (see


Multiplexing (pooling) strategies on page 27).
If overall concentration is too high, dilute pooled
samples with deionized water before PCR.
Increasing the MgCl2 concentration of some
samples can reduce the disparity in peak heights,
but may also increase the amplification of
non-specific products (background).

Sudden loss of signal in all


samples

Instrument laser power or


current problem.

Check laser power and current in the EPT window


(see Examine the sample info, raw data, and EPT
trace on page 154).

Multiple dye colors are detected


as one dye color

5-dye samples were run with a


4-dye matrix.

Repeat run with correct dye set.

DNA Fragment Analysis by Capillary Electrophoresis

171

11

Chapter11 Troubleshooting
Extra peaks troubleshooting

Extra peaks troubleshooting


Symptom
Extra peaks

Possible cause

Action

Pull-up or cross-talk due to


saturated data in a dye color (for
example, a high intensity blue
peak can create pull up peaks in
other colors). See Signal
intensity is too high or
saturated on page 166.

Decrease sample concentration during PCR or


when preparing samples for electrophoresis.

Degraded PCR products.

Repeat PCR.

Stutter peaks.

See Identifying stutter products in microsatellite


analysis on page 113.

Incomplete restriction or
ligation (AFLP applications only).

Extract the DNA again and repeat the


restriction-ligation.

Sample is not denatured.

Make sure the samples are heated at 95C for


3 to 5 minutes, then immediately placed on ice for
2 to 3 minutes before loading.

Hairpin secondary structures


are present in PCR primers.

See PCR troubleshooting on page 176.

Non-specific primer peaks.

172

Primer/dimer peaks.

See Primer design guidelines on page 29.

Artifact peaks.

See Pull-up peaks from a sample appear in the


red or orange dye signal, and are detected as
size-standard peaks due to over-saturation of
sample-peak signal. on page 185.

Sample or reagent
contamination.

Use fresh sample or reagent.

Contamination with exogenous


DNA.

Use appropriate techniques to avoid introducing


foreign DNA during laboratory handling.

Renaturation of denatured
samples.

Load the sample immediately following


denaturation, or store it on ice until ready.

DNA Fragment Analysis by Capillary Electrophoresis

Chapter11 Troubleshooting
Extra peaks troubleshooting

Symptom

Possible cause

11

Action

Pull-up peaks

Incorrect or poor-quality matrix


or spectral calibration.

Run a new spectral calibration.

Wrong matrix or spectral use for


analysis of 310 instrument data.

Reanalyze with the correct matrix in the


GeneMapper Software.

Offscale, saturated signal in


primary peak caused by high
sample concentration.

Decrease sample concentration.

Polymer on instrument >7 days,


degraded polymer
contaminants.

Perform warm water wash(es) and replace


polymer.

DNA Fragment Analysis by Capillary Electrophoresis

Edit the instrument protocol to specify the correct


spectral calibration.

Decrease injection time.

If the problem persists, replace the capillary array.

173

11

Chapter11 Troubleshooting
Extra peaks troubleshooting

Symptom

Possible cause

Action

Data spikes

Bubbles, dried polymer, or dust


in the capillary array migrate
past the camera.

1. Flush the water trap. Refer to the instrument


user guide for information.
2. Check for bubbles and run the bubble wizard if
any are visible. Clean all connections and tubing
around the instrument pump.
3. Check the polymer bottle, the area around the
pump lines, and the array port for crystals. If
present, warm the polymer gently to 30C with
gentle mixing, then refill the pump and array
with the polymer.
4. If the problem persists, perform a water wash
and replace the polymer.

174

DNA Fragment Analysis by Capillary Electrophoresis

Chapter11 Troubleshooting
Extra peaks troubleshooting

Symptom

Possible cause

11

Action

Split peaks

Plus A and/or minus A peaks


due to:

Repeat the experiment with a lower initial


template concentration.

Too many fragments for


A-addition.

Modify the experiment to:

Suboptimal PCR conditions


and/or suboptimal primer
design.

Increase addition of A:
Add a final extension step of 60 minutes at
72C.
Increase Mg2+ concentration
Use ABI PRISM Tailed Primer Pairs
Remove A by enzymatic treatment (T4 DNA
polymerase)
For more information, see Avoiding incomplete 3'
A nucleotide addition on page 34.

Many small extraneous peaks


appearing next to a
high-intensity peak

Background signal is above


Minimum Peak Height value.

Adjust the setting in analysis method.

High sample concentration.


(Extraneous peaks represent
non-specific DNA comigrating
with main fragment peak.)

Dilute the sample.

Sample concentration is too


high.

Decrease the injection time or injection voltage.


See Optimizing electrokinetic injection
parameters on page 78.

DNA Fragment Analysis by Capillary Electrophoresis

175

11

Chapter11 Troubleshooting
PCR troubleshooting

PCR troubleshooting
Symptom
Poor priming resulting in
weak signal

Possible Cause
Melting temperature is too
low due to low G+C content
and/or short primer length.

Action
Evaluate primer design.

Secondary structure of the


primer, particularly at the 3'
end.
Secondary structure of the
template in the region of
hybridization.

Amplified DNA
concentration is lower than
expected

PCR inhibition

Insufficient [F]dNTPs added


to PCR reaction.

Reamplify using more [F]dNTPs or examine the efficiency


of the PCR.

Amplification cycle setting is


too low.

Add 3 to 5 cycles.

Low MgCl2 concentration.

Increase the MgCl2 concentration.

Low affinity of the primer to


the template.

Decrease the annealing temperature 2 to 3C at a time.


Background signal may increase.

Low sample concentration.

Increase sample concentration.

Inhibitors in template.

Purify template (see Purifying DNA on page 56).

Thermal cycler malfunction.

Troubleshoot the thermal cycler problem. Refer to the


thermal cycler user guide for information.

PCR reagents are


contaminated or expired.

Use fresh PCR reagents.

Degraded primers.

Store unused primers at 15 to 25C. Do not expose


fluorescent dye-labeled primers to light for long periods of
time.

Sample contains
hemoglobin, heparin,
polyphenol (plant),
polysaccharides.

Dilute the sample before amplification to reduce the


amount of PCR inhibitors.

Extraction introduced
inhibitors (chloroform,
phenol, EDTA, detergents
(SDS), xylol, ethanol,
bromophenol blue).
Contamination with
exogenous DNA

176

Carryover.

Use appropriate techniques to avoid introducing foreign


DNA during laboratory handling. For more information,
see Avoiding contamination on page 64,

DNA Fragment Analysis by Capillary Electrophoresis

Chapter11 Troubleshooting
PCR troubleshooting

Symptom
Poor amplification,
non-specific amplification

Possible Cause
Poor-quality or degraded
DNA template.

11

Action
Use fresh template.
Run an agarose gel to check sample concentration and
quality.
If DNA is stored in water, check water purity.

Poor amplification, nonspecific amplification in


restriction-ligation
experiments (AFLP only)

Insufficient or excess
template DNA.

Use recommended amount of template DNA. Run an


agarose gel to check sample concentration and quality.

PCR inhibitors in the DNA


sample (binding proteins,
salts that carry over from
poor DNA extractions).

Use different extraction procedures.

Incorrect thermal cycling


parameters.

Check protocol for correct thermal cycling parameters.

Incorrect pH.

Use correct concentration of DNA and buffer.

Tubes loose in the thermal


cycler.

Push reaction tubes firmly into contact with block before


first cycle.

Third-party or non-PCR tube


type used.

Use GeneAmp Thin-Walled Reaction Tubes with Caps


with Thermo Fisher Scientific thermal cyclers.

Primer concentration too


low.

Use recommended primer concentration.

Primer design.

See Non-specific amplification on page 32.

Incomplete restrictionligation (in experiments


involving restriction-ligation)
due to insufficient or
insufficiently active ligase.

1. Test the ligase activity with control DNA.


2. Repeat restriction-ligation with a higher concentration
of ligase (in Weiss units).
Note: 1 Weiss unit = 67 cohesive-end ligation units
If the problem persists, repeat the restriction-ligation
with fresh enzymes and buffer. Use an agarose gel to
check the reaction results.
Refer to the AFLP Plant and Microbial Protocols for
more information (see AFLP applications on
page 200).

TE0.1 is buffer not properly


made, or contains too much
EDTA.

Add the appropriate amount of MgCl2 solution to amplified


reaction. Remake the TE0.1 buffer.

Insufficient enzyme activity.

Repeat the experiment with the recommended amount of


restriction enzyme, ligase, and AmpliTaq DNA
Polymerase.
Note: 1 Weiss unit = 67 cohesive-end ligation units.

Hairpin secondary
structures in PCR primers

Primer design.

DNA Fragment Analysis by Capillary Electrophoresis

See Primer design guidelines on page 29.

177

11

Chapter11 Troubleshooting
Irregular baseline troubleshooting

Symptom

Possible Cause

Primer/dimer formation

MgCl2 concentration.

Action
See Optimizing PCR on page 55.

Annealing temperature in
the PCR.
Primer design.

See Primer design guidelines on page 29.

Too much primer added to


reaction.

Prepare new reaction.

Primer over-amplification
due to insufficient or
poor-quality template.

Prepare new reaction.

Irregular baseline troubleshooting


Excessivenoiseoranelevatedbaselineaffectsbothsizingandgenotypingresults.
Symptom
Constant elevated signal
in raw data

Possible Cause

Action

Baseline waterfall

Waterfall (most
common on 310
instruments)

Contamination from marker


pen ink (if you used a marker
to label the plate or the heat
seal).

178

Prepare new plate.

DNA Fragment Analysis by Capillary Electrophoresis

Chapter11 Troubleshooting
Irregular baseline troubleshooting

Symptom
(continued)
Constant elevated signal
in raw data
Waterfall (most
common on 310
instruments)

Possible Cause

11

Action

Contamination from water


used to make buffer, wash
reservoirs/septa, for sample
injection, for any sampleprep steps, or for water
wash.

Use fresh water.

Instrument contamination.

Refer to the instrument user guide for troubleshooting


information.

Improperly filled/leaky
connections, tubing, or
polymer block.
Spectral/matrix calibration
issue.

Use correct matrix standard (see Dye sets and matrix


standards on page 41).
Specify the correct dye set in the instrument protocol.
Ensure the correct dye set was selected in spectral
calibration.
Apply the correct matrix file (310 instruments only).

Noisy baseline

Polymer on instrument
>7 days, polymer degraded
or precipitated.

Perform warm water wash(es) and replace polymer. If the


problem persists, replace the array.

Arcing/electronic noise.

Remove bubbles. Refer to the instrument user guide for


information.

Amplification of non-specific
products during PCR.

See PCR troubleshooting on page 176.

Degraded or incorrectly
stored Hi-Di Formamide
can cause low signal and
degraded products.

Use fresh, properly stored Hi-Di Formamide. See HiDi Formamide storage on page 82.

Capillary is contaminated.

Perform a water wash.

Weak or low signals and/or


an elevated baseline.

See Irregular signal intensity troubleshooting on


page 164.

High salt concentration.

Salt preferentially injects smaller fragments and inhibits


injection of larger fragments, so the majority of salt may
have been injected in the first injection.
Re-inject the sample.
If signal intensity does not increase, see Desalting on
page 190.

Adequate signal strength


with noisy data

Electrical noise

Contact Thermo Fisher Scientific.

Secondary hybridization site


is present on primer, which
results in many extra peaks.

Evaluate primer design.

Impure primer. You may see


a shadow sequence of N-1.

HPLC-purify the primer.

DNA Fragment Analysis by Capillary Electrophoresis

179

11

Chapter11 Troubleshooting
Instrumentation troubleshooting

Instrumentation troubleshooting
Somedataqualityissuesarenotsamplerelated,butarecausedbysettingsor
conditionsusedfortheinstrumentrun.
Note: ThecolorofthecurrenttracevariesbetweenversionsofDataCollection
Software.
Symptom
Low or fluctuating current

Possible Cause

Action

Expired or incorrectly stored


buffer and/or polymer.

Use fresh buffer and polymer.

Bubbles in polymer.

Remove bubbles. Refer to the instrument user guide for


information.

Anode buffer jar (3130


Series, 3730 Series, or 310
instruments) buffer is not
above required level.

Fill the anode buffer jar to the required level.

ABC (3500 Series


instruments) buffer is not
above required level.

For opened containers: Pipet buffer from the overflow


chamber to the main chamber.

Fluctuating current

Arcing caused by bubbles.

Remove bubbles. Refer to the instrument user guide for


information.

Current too high

Decomposition of urea in the


polymer.

Use fresh polymer.

Incorrect buffer formulation


(most likely too
concentrated) (not applicable
to 3500 Series instruments).

Use correctly prepared buffer.

Arcing to conductive surface


on the instrument.

Ensure that the ambient temperature is 15 to 30C and the


humidity is <80%. Check for excessive condensation on the
instrument.

180

For unopened containers: Invert the


ABC, then tilt it slightly to make
sure most of the buffer is in the
larger side of the container. There
should be less than 1 mL of the
buffer remaining in the smaller side
of the container.

DNA Fragment Analysis by Capillary Electrophoresis

Chapter11 Troubleshooting
Instrumentation troubleshooting

Symptom
Drop-off of current signal

Possible Cause

11

Action

Data Collection Software EPT view (current trace is blue)

Current signal should be


horizontal

Instrument has stopped


running and red light is on.
There are black marks
inside the instrument.

Air bubble in lower polymer


block.

Remove bubbles. Refer to the instrument user guide for


information.

Clogged capillary (caused by


sample overloading).

Flush the capillary array with polymer.

Arcing to conductive surface


on the instrument.

Ensure that the ambient temperature is 15 to 30C and the


humidity is <80%. Check for excessive condensation on the
instrument.

Arcing caused by bubbles in


polymer.

Remove bubbles from polymer.

GeneMapper Software EPT view (current trace is green)

Current signal shape should resemble


the example shown in Examine the
sample info, raw data, and EPT trace on
page 154

DNA Fragment Analysis by Capillary Electrophoresis

181

11

Chapter11 Troubleshooting
Sizing or Size Quality (SQ) troubleshooting

Sizing or Size Quality (SQ) troubleshooting


Viewing the
size-standard
definition

Sizingissuescanoccurifthepeaksdetecteddonotmatchthepeakslistedinthe
sizestandarddefinition,forexample,ifadditionalpeaksaredetectedassizestandard
peaks,orifsizestandardpeaksarenotdetected.
Toviewthepeaksdetectedinthesizestandardandthepeakslistedinthe
sizestandarddefinitionforasample,selectthesample,thenclick (AnalysisSize
MatchEditor)toviewthesizinginformationfortheselectedsample(s).

Iftheexpectedpeaksarenotdetected,yourfirstcourseofactionshouldbeto
determinethecauseofthepeakdetectionissueandresolvetheissue.
Ifyouwanttousethedataevenifthesizestandarddataisoflowerquality,youcan
modifythesizestandarddefinition(describedbelow)toeliminateoraddpeaksto
improvethesizestandardqualityresult.
Note: Datafor

SQsamplesisviewableonlyintheRawDataview.

FormoreinformationontechniquesforimprovingsizingaccuracyonThermoFisher
Scientificgeneticanalyzers,refertoRosenblumetal.(1997)andWenzetal.(1998).

Modifying the
size-standard
definition

1. IntheProjectWindow,selectToolsGeneMapperManager.
2. SelecttheSizeStandardtab,thenselectthesizestandarddefinitionusedto
analyzethedata.

3. SelectSaveAsandnamethenewsizestandarddefinition.

182

DNA Fragment Analysis by Capillary Electrophoresis

Chapter11 Troubleshooting
Sizing or Size Quality (SQ) troubleshooting

11

4. Openthenewsizestandarddefinitionandaddorremovepeaksasneeded.

Troubleshooting
Symptom
SQ

or

and...

Possible Cause

No sample plot is displayed for a


sample with the error No sizing
data

A plot is not displayed if sizing


fails.

Size Match Editor does not


display peak data

Incorrect Size Standard Dye


specified in size-standard
definition.

Missing size-standard peaks

The fragment sizes of the sizestandard definition do not match


the positions of the detected
peaks.

Action
See Sizing or Size Quality (SQ) troubleshooting
on page 182.
Note: You can view data for failed sizing in the Raw
view (see Examine the sample info, raw data, and
EPT trace on page 154).

The Peak Amplitude Threshold


of the dye color associated with
the size standard is set too high
or low in the analysis method.

Verify that the correct dye and fragment sizes are


specified in the Size Standard definition.
Select, modify, or create correct size-standard
definition as needed (see Modifying the
size-standard definition on page 182).

Adjust the analysis method so that the peak


detection threshold associated is greater than the
height of the noise signal. See GeneMapper
Software peak detection settings on page 94.
IMPORTANT! We do not recommend decreasing
the threshold below 50 RFU.

Expired or degraded size


standard.

DNA Fragment Analysis by Capillary Electrophoresis

Use fresh size standard.

183

11

Chapter11 Troubleshooting
Sizing or Size Quality (SQ) troubleshooting

Symptom
SQ

or

and...

Missing size-standard peaks


(continued)

Possible Cause

Action

Incorrect concentration of size


standard in sample loading
reagent.

Increase the concentration of size standard added


to subsequent runs.

Size-standard peaks are not


migrating as expected during a
normal run time.

See Migration troubleshooting on page 168.

Incorrect injection settings (for


example, the injection time is
too short).

Review the injection settings of the run module for


errors.

High salt concentration.

Salt preferentially injects smaller fragments and


inhibits injection of larger fragments, so the
majority of salt may have been injected in the first
injection.
Re-inject the sample.
If signal intensity does not increase, see
Desalting on page 190.

Smaller size-standard
fragments are not labeled

Size standard peak and primer


peak are in the same read
region (see figure below).

Modify the size standard-definition and remove the


size-standard peak that overlaps with the primer
peak.
Change the analysis range in the Advanced Peak
Detection Algorithm field in the Peak Detector tab
of the analysis method. Select Partial Range and
select a starting data point that eliminates the
primer peaks from analysis.

Size-standard peaks labeled in


primer read region

Larger size-standard peaks are


not present in trace

184

Size-standard definition
modified to eliminate peaks
labeled in primer read region

Run time too short.

Increase run time.

Late start caused by a reagent


issue, a blocked capillary, or
high sample concentration.

Use fresh polymer.

A non-Thermo Fisher Scientific


size standard was used.

Use a Thermo Fisher Scientific size standard.

DNA Fragment Analysis by Capillary Electrophoresis

Chapter11 Troubleshooting
Sizing or Size Quality (SQ) troubleshooting

Symptom
SQ

or

and...

Possible Cause

11

Action

Extra peaks in size-standard


trace

Pull-up peaks from a sample


appear in the red or orange dye
signal, and are detected as
size-standard peaks due to
over-saturation of sample-peak
signal.

Decrease sample concentration.

Spectral calibration performed


with the incorrect matrix
standard for the dye set.

Perform a spectral calibration with the correct


matrix standard for the dye set (see Table18 on
page 86).

Spectral calibration is from a


different array or has not been
run within the last 3 months.

If the peak heights that cause the pull-up peak are


not near the saturation limits of the instrument,
repeat the spectral calibration.

The signal in a neighboring


capillary is very strong and
creating a bleed-through peak.

Decrease the sample concentration.

Degraded size standard. A size


standard can be degraded by
sitting at room temperature for
>24 hours or using improperly
stored Hi-Di Formamide.

Use fresh size standard and fresh, properly stored


Hi-Di Formamide. See Hi-Di Formamide
storage on page 82.

Wrong size-standard definition


was used for the analysis.

Re-analyze with correct size-standard definition.

Degraded or incorrectly stored


Hi-Di Formamide.

Use fresh, properly stored Hi-Di Formamide. See


Hi-Di Formamide storage on page 82.

DNA Fragment Analysis by Capillary Electrophoresis

Decrease injection time.

Decrease the injection time.

185

11

Chapter11 Troubleshooting
Sizing or Size Quality (SQ) troubleshooting

Symptom
SQ

or

and...

Sizing failures occur in a regular


pattern (the same wells fail
repeatedly)

Possible Cause

Action

Electrophoresis or pipetting
error.

Refer to the instrument user guide for information


on troubleshooting capillaries/arrays.

Defective capillaries/arrays.
Autosampler is misaligned.

Noise peaks are detected as


size-standard peaks

Noise
peaks

Contaminated sample.

Prepare new sample.


Use fresh, properly stored Hi-Di Formamide. See
Hi-Di Formamide storage on page 82.

The Peak Amplitude Threshold


of the dye color associated with
the size standard is set too high
or low in the analysis method.

Adjust the analysis method so that the peak


detection threshold associated is greater than the
height of the noise signal. See GeneMapper
Software peak detection settings on page 94.
Note: You can adjust the threshold if you want to
examine the data. However, we recommend that
you rerun with increased size-standard
concentration.

Size call inaccurate for known


DNA sample

186

Incorrect size standard was


added to sample.

Repeat with the correct size standard.

Size standard peaks are not


sized correctly.

Examine the size standard peaks and compare the


sizes to the size standard definition. See Viewing
the size-standard definition on page 182.

Migration issues.

See Migration troubleshooting on page 168.

DNA Fragment Analysis by Capillary Electrophoresis

Chapter11 Troubleshooting
GeneMapper Software troubleshooting

11

GeneMapper Software troubleshooting


Someproblemswithdatacanbecausedbythesettingsusedtoanalyzethedata.If
peakheight,morphology,andnumberofexpectedpeaksareacceptable,butthe
samplefailssizing,itmaybecausedbyanalysismethodsettingsthatarenot
optimizedforyourapplication.
Foradditionaltroubleshootinginformation,refertotheGeneMapperSoftwareHelp
andtheGeneMapperSoftwareReferenceandTroubleshootingGuide(Pub.no.4403673).
Symptom

Possible Cause

Action

Generic message - the error


for each sample may be
different.

View the Info tab (see Examine the sample info, raw data,
and EPT trace on page 154).

The bin set in the analysis


method does not match the
panel used for analysis.

Modify the Bin Set selection on the Allele tab in the


analysis method.

GeneMapper Software
error message

Error Message: The bin set


in the analysis method does
not match the panel used for
analysis.

DNA Fragment Analysis by Capillary Electrophoresis

For information on resolving the error, refer to the


GeneMapper Software Reference and Troubleshooting
Guide.

187

11

Chapter11 Troubleshooting
GeneMapper Software troubleshooting

Symptom

Possible Cause

Action

al? label
or alleles are not falling
within bins

Allele not labeled

Allele is migrating at a
different rate than expected.

See Migration troubleshooting on page 168.

Bin is not defined for the


allele.

Modify the binset to define the allele.

Peak is detected as stutter.

Decrease the Stutter ratio in the analysis method.


If the problem persists, mask the stutter peak by labeling
the allele as a mono peak: In the Panel Manager, change
the marker repeat # to 1 for the marker in question.

Data not sorted by name

Peak intensity is below the


Peak Amplitude Threshold in
the analysis method.

Adjust the Peak Amplitude Threshold in the analysis


method.

The default setting in


GeneMapper Software
sorts the data by status
rather than sample name.

To resort the data:


Select EditSort, or
Shift+click the column header to sort by
Clicking once sorts in ascending order, clicking twice sorts
in descending order.

When adding samples to a


project, the expected data
files are not listed in the Add
Samples to Project dialog
box

Data was not collected as


expected.

Ensure the following on the Data Collection Software


computer:
The instrument completed the run(s) and data are
visible in Data Collection Software.
Sample files were extracted successfully (3130 Series
or 3730 Series instruments).
The run folder was created and saved on the
instrument computer.
The correct number of *.fsa sample files were created
within the run folder.

188

DNA Fragment Analysis by Capillary Electrophoresis

Chapter11 Troubleshooting
GeneMapper Software troubleshooting

Symptom

Possible Cause

11

Action

Genotypes tab is grayed

No panel specified before


analysis.

Specify a panel in the Project window before analysis.

The software is detecting


two separate peaks as one.

Adjust analysis method Peak Detector settings:

Two peaks do not separate


and are detected as one
peak

Polynomial degree:
A higher setting increases the sensitivity of the curvefitting process. A lower setting decreases it.
Peak Window size value:
A lower setting increases the sensitivity of the curvefitting process. A higher setting decreases it.
Decrease the peak window size to 13 and re-analyze.
For more information, see GeneMapper Software peak
detection settings on page 94.

DNA Fragment Analysis by Capillary Electrophoresis

189

11

Chapter11 Troubleshooting
Preamplification gel troubleshooting

Preamplification gel troubleshooting


Symptom

Possible Cause

No DNA smear on agarose


gel (AFLP only)

Incomplete digestion of
DNA.

Action
Check that enzymes are not expired, incubation
temperature settings are correct, and so on. Redigest
DNA.

Desalting
Impact of high salt
concentration

Sampleshighinsaltresultinpoorinjectionsandlowsignalintensity.Youmaybeable
tocompensatefordecreasedsignalintensityby:
Reinjectingthesample.Saltpreferentiallyinjectssmallerfragmentsandinhibits
injectionoflargerfragments,sothemajorityofsaltmayhavebeeninjectedinthe
firstinjection.
Increasingtheinjectiontimeand/orinjectionvoltage.
Ifreinjectingandincreasingtheinjectionparametersdoesnotimprovesignalintensity,
desaltand/orconcentratethesamples.
Donotincreasesampleconcentrationbyevaporatingthesampleswithoutperforming
adesaltingstep.Doingsoincreasesthesaltconcentrationandpreventscomplete
denaturationoftheDNAwhichcausesdecreasedsignalstrength.

Eliminating salt
concentration as
the cause

Todetermineifsaltconcentrationiscausinglowsignalintensity,runthesizestandard
alone(seeRunningcontrolstoisolateaproblemonpage 156).Ifyousee:
Resolutionlosswiththesizestandard,troubleshootinstrument/reagentissue.
Refertotheinstrumentuserguideforinformation.
Noresolutionlosswiththesizestandard,addsampletothewellcontainingthe
sizestandardandrunagain.Ifresolutiondecreases,desaltthesample.

Desalting

Todesaltyoursample(s),tryoneofthefollowing:
NucAwaySpinColumnsNucAwaySpinColumnsremoveunincorporated
nucleotidesandsaltsafterprobesynthesisreactions.Rehydratethecolumn,
centrifugetoremovetheinterstitialfluid,addthesampletothetopofthecolumn,
andcentrifugeagain.
AmiconCentricon100Microconcentrator(orCentricon30forfragmentssmaller
than130bp)
EthanolprecipitationofthepooledPCRproduct,followedbyresuspensionin
distilled,deionizedwater.RefertoMolecularcloning(Sambrook,Fritsch,and
Maniatis1989)forprotocols.
Sampledialysisonafiltermembrane:
a. FloataMilliporeVSfilter(MilliporePartno.VSWP02500),shinysideup,
ontopof50mLofdeionized,autoclavedwaterina50mLconicalplastic
tube.
b. Carefullyspot~15Lofsampleontopofthefilter,usinganappropriate
pipette.

190

DNA Fragment Analysis by Capillary Electrophoresis

Chapter11 Troubleshooting
Evaluating 310 Genetic Analyzer multicomponent matrix quality

11

c. Dialyzethesamplefor20minutes.
d. Usingapipette,verycarefullyremovethesampleanddilute.

Evaluating 310 Genetic Analyzer multicomponent matrix quality


Purpose of the
multicomponent
matrix
Factors affecting
matrix quality

Themulticomponentmatrixcompensatesfortheoverlapofdifferentdyecolorsby
subtractingout,ineachdyesdetectionrange,theportionofthesignaldueto
fluorescencefromotherdyes.
Reagentquality/freshness
Buffertypeandconcentration
Polymertype
Denaturingornondenaturingconditions
Runtemperature

When to create a
new matrix

Whenyoucreateamatrix,youmustruneachdyematrixstandardseparatelyto
determinetheproportionalamountoffluorescencethatisemittedinallfourdetection
regions.
Becausetheemissionspectraofthedyesvarywiththephysicalenvironment(suchas
thepHorpolymertypeandconcentration),createanewmatrixifrunconditions
change.
Ifyouobserveanyofthesymptomslistedinthetableonthenextpage,createanew
matrix.Youcanapplythenewmatrixtooldsamplesandreanalyzethedata.

Virtual Filter Set C

MatrixfilesmadeforVirtualFilterSetCareespeciallysusceptibletominorchangesin
runconditionsbecauseoftheemissionmaximumof6FAMdye(therecommended
bluedisplayingdyeforthisfilterset):
Itisveryclosetothelaserwavelengthof514.5nm.Thus,thewindowforcollected
bluelightintensitydataisoffsettolongerwavelengthsanddoesnotcontainthe
emissionmaximumof6FAMdye.
ItisalsoveryclosetothedetectionregionforthegreendisplayingTETdye.
IfyouareusingVirtualFilterSetC,watchforevidenceofmatrixproblemsandcreate
anewmatrixassoonasproblemsappear

Identifying matrix
problems

Apoororincorrectmatrixresultsintoomuchortoolittlesubtractionofdyespectral
overlapduringdataanalysis.Eachcausesarecognizableelectropherogramanomaly:
Bleedthroughpeaks,alsocalledpullups,arecausedbytoolittlesubtraction
Bleedthroughpeaksaresmallpeaksofonecolorlyingdirectlyunderalargepeak
ofanothercoloreventhoughthereisnoPCRproductcorrespondingtothe
smallerpeak.
Elevatedinterpeakbaselineiscausedbytoomuchsubtraction
Thetableonthenextpagecontainsexamplesofthesymptomslistedabove.

DNA Fragment Analysis by Capillary Electrophoresis

191

11

Chapter11 Troubleshooting
Evaluating 310 Genetic Analyzer multicomponent matrix quality

Symptom

Possible Cause

Action

Pull-up peaks (too little


matrix subtraction)

The matrix was made with the wrong


dyes or filter set.

Create a new matrix with the correct dye and


filter set. See Dye sets on page 41.

The signal from a large peak is off-scale


because of sample overloading. In the
raw data, the peak showing pull-up is
off-scale.

Keep peak heights between approximately 150


and 4000 RFU. If sample data is off-scale, do
one of the following:
Rerun the samples using a shorter injection
time.
Dilute and rerun the samples.
Create a new matrix with the correct dye
and filter set. See Dye sets on page 41.

Elevated interpeak baseline


(too much matrix
subtraction)

Too much matrix subtraction

192

Create a new matrix.

DNA Fragment Analysis by Capillary Electrophoresis

Ordering Information

Thermalcyclersandaccessories........................................ 193

Geneticanalyzersandconsumables .................................... 194

GeneScansizestandards ............................................ 197

Matrixstandardsforspectralcalibration ................................ 197

Installationstandards................................................. 197

Reagentkits ......................................................... 198

Otherusersuppliedmaterials ......................................... 198

Thermal cyclers and accessories


Item
Veriti 96-Well Thermal Cycler
GeneAmp PCR System 9700 with the Silver 96-Well Block
GeneAmp PCR System 9700 with the Gold-plated Silver 96-Well Block
Silver 96-Well Sample Block
Gold-plated Silver 96-Well Sample Block

Source
4375786
N8050001
4314878
N8050251
4314443

MicroAmp

Autoclaved Reaction Tubes with Caps, 0.2 mL

N8010612

GeneAmp

Autoclaved Thin-walled Reaction Tubes with Domed Caps, 2000 tubes

N8010537

GeneAmp

Autoclaved Thin-walled Reaction Tubes with Domed Caps, 1000 tubes

N8010611

MicroAmp 96-Well Tray

N8010541

MicroAmp Reaction Tube with Cap, 0.2-mL

N8010540

MicroAmp 8-Tube Strip, 0.2-mL

N8010580

MicroAmp

8-Cap Strip

N8010535

MicroAmp

96-Well Tray/Retainer Set

MicroAmp

96-Well Base

MicroAmp

Clear Adhesive Film

4306311

MicroAmp

Optical Adhesive Film

4311971

MicroAmp Optical 96-Well Reaction Plate

DNA Fragment Analysis by Capillary Electrophoresis

403081
N8010531

N8010560

193

Ordering Information
Genetic analyzers and consumables

Genetic analyzers and consumables


Item

Source

Applied Biosystems 3500 or 3500xL Genetic Analyzer


Applied

Biosystems

Contact your local


Thermo Fisher Scientific
sales representative

3500xL Genetic Analyzer

3130 or 3130xl Genetic Analyzer


3730 or 3730xl DNA Analyzer
310 Genetic Analyzer
3500/3500xL Analyzer materials
Anode Buffer Container (ABC)

4393927

Cathode Buffer Container (CBC)

4408256

Septa Cathode Buffer Container, 3500 Series

4410715

Conditioning Reagent

4393718

Capillary Array, 36 cm, 8 Capillary, 3500 Genetic Analyzers

4404683

Capillary Array, 50 cm, 8 Capillary, 3500 Genetic Analyzers

4404685

Capillary Array, 36 cm, 24 Capillary, 3500xL Genetic Analyzers

4404687

Capillary Array, 50 cm, 24 Capillary, 3500xL Genetic Analyzers

4404689

GeneScan Size Standards

See GeneScan size


standards on page 197.

Matrix Standard Kits

See Matrix standards


for spectral calibration
on page 197.

POP-4 Polymer (960 Samples), 3500/3500xL Genetic Analyzers

4393710

POP-4 Polymer (384 Samples), 3500/3500xL Genetic Analyzers

4393715

POP-6 Polymer (960 Samples), 3500/3500xL Genetic Analyzers

4393712

POP-6 Polymer (384 Samples), 3500/3500xL Genetic Analyzers

4393717

POP-7

Polymer (960 Samples), 3500/3500xL Genetic Analyzers

4393710

POP-7

Polymer (384 Samples), 3500/3500xL Genetic Analyzers

4393715

8-Tube Retainer and Base Set (Standard) for 3500/3500xL Genetic Analyzers

4410231

8-Strip Septa for 3500/3500xL Genetic Analyzers

4410701

96-well Retainer and Base Set (Standard) 3500/3500xL Genetic Analyzers

4410228

96-Well Septa for 3500/3500xL Genetic Analyzers

4412614

3730/3730xl Analyzer materials


Capillary Array, 50 cm, 48 Capillary, 3730 for Genetic Analyzers

4331250

Capillary Array, 36 cm, 48 Capillary, 3730 for Genetic Analyzers

4331247

Capillary Array, 50 cm, 96 Capillary, 3730xl for Genetic Analyzers

4331246

Capillary Array, 36 cm, 96 Capillary, 3730xl for Genetic Analyzers

4331244

GeneScan

194

Size Standards

See GeneScan size


standards on page 197.

DNA Fragment Analysis by Capillary Electrophoresis

Ordering Information
Genetic analyzers and consumables

Item
Matrix Standard Kits

Source
See Matrix standards
for spectral calibration
on page 197.

POP-7 Polymer (28 mL), 3730/3730xl Genetic Analyzers

4363929

POP-7

Polymer (140 mL), 3730/3730xl Genetic Analyzers

4335615

POP-7

Polymer (280 mL), 3730/3730xl Genetic Analyzers

4363935

POP-7 Polymer (840 mL), 3730/3730xl Genetic Analyzers

4335611

MicroAmp Optical 96-Well Reaction Plate

N8010560

Hi-Di Formamide

4311320

Reservoir Septa

4315932

Running Buffer, 10

402824

96-Well Plate Septa

4315933

3130/3130xl Analyzer materials


96-Well Plate Septa

4315933

Reservoir Septa

4315932

Capillary Array, 80 cm, 4 Capillary, 3130 Genetic Analyzers

4333465

Capillary Array, 50 cm, 4 Capillary, 3130 Genetic Analyzers

4333466

Capillary Array, 36 cm, 4 Capillary, 3130 Genetic Analyzers

4333464

Capillary Array, 22 cm, 4 Capillary, 3130 Genetic Analyzers

4333463

Capillary Array, 80 cm, 16 Capillary, 3130xl Genetic Analyzers

4319899

Capillary Array, 50 cm, 16 Capillary, 3130xl Genetic Analyzers

4315930

Capillary Array, 36 cm, 16 Capillary, 3130xl Genetic Analyzers

4315931

Capillary Array, 22cm, 16 Capillary, 3130xl Genetic Analyzers

4319898

GeneScan Size Standards

See GeneScan size


standards on page 197.

Matrix Standard Kits

See Matrix standards


for spectral calibration
on page 197.

POP-4 Polymer (7000 L), 3130/3300xl Genetic Analyzers

4352755

POP-4 Polymer (3500 L), 3130/3300xl Genetic Analyzers

4363752

POP-6 Polymer (7000 L), 3130/3300xl Genetic Analyzers

4352757

POP-6

Polymer (3500 L), 3130/3300xl Genetic Analyzers

4363783

POP-7

Polymer (7000 L), 3130/3300xl Genetic Analyzers

4352759

POP-7

Polymer (3500 L), 3130/3300xl Genetic Analyzers

4363785

Running Buffer, 10
MicroAmp

Optical 96-Well Reaction Plate

402824
N8010560

3100/3100-Avant Genetic Analyzer Autosampler Plate Kit, 96-well

4316471

Hi-Di Formamide

4311320

310 Analyzer materials

DNA Fragment Analysis by Capillary Electrophoresis

195

Ordering Information
Genetic analyzers and consumables

Item

Source

Capillary Array, 47-cm, 310 DNA Analyzer capillary array

402839

Capillary Array, 61-cm, 310 DNA Analyzer capillary array

402840

96-well tray adaptor (for 9700 thermal cycler trays)

4305051

GeneScan Size Standards

See GeneScan size


standards on page 197.

Matrix Standard Kits

See Matrix standards


for spectral calibration
on page 197.

0.5 mL sample tray

5572

MicroAmp 8-tube strip, 0.2-mL

N8010580

MicroAmp 96-well base (holds 0.2-mL reaction tubes)

N8010531

MicroAmp

96-well full plate cover

N8010550

MicroAmp

96-well tray/retainer set

403081

POP-4

Polymer (5000 L), 310 Genetic Analyzers

402838

POP-6

Polymer (5000 L), 310 Genetic Analyzer

402837

Hi-Di

Formamide

4311320

Running Buffer, 10

4335643

Genetic Analyzer Septa Retainer Clips for 96-tube Sample Tray

402866

Genetic Analysis Sample Tubes (0.5-mL)

401957

Septa for 0.5-mL Sample Tubes

401956

For the Safety Data Sheet (SDS) of any chemical not distributed by Thermo Fisher Scientific, contact the chemical manufacturer. Before handling
any chemicals, refer to the SDS provided by the manufacturer, and observe all relevant precautions.

196

DNA Fragment Analysis by Capillary Electrophoresis

Ordering Information
GeneScan size standards

GeneScan size standards


LIZ dye-labeled size
standards
(5-dye chemistry)

ROX dye-labeled size


standards
(4-dye chemistry)

Part no.

Part no.

GeneScan 120 LIZ

4324287

GeneScan 350 ROX

401735

GeneScan 500 LIZ

4322682

GeneScan 400HD ROX

402985

GeneScan 600 LIZ

4366589

GeneScan 500 ROX

401734

4408399

GeneScan

401098

GeneScan
GeneScan

600

LIZ

1200

v2.0

LIZ

1000

4379950

ROX

For denaturing and non-denaturing applications.


For non-denaturing applications only.

Matrix standards for spectral calibration


Dyesetshavebeentestedandoptimizedfortheinstrument,exceptwherenoted.
Dye Set part numbers
Instruments
DS-02

DS-20

DS-30

DS-31

DS-32

DS-33

DS-34

3500 Series

4323014

Not
tested

4345827

4345829

4345831

4345833

Not
tested

3730 Series

4323014

Not
tested

4345827

4345829

4345831

4345833

Not
tested

3130 Series

4323014

Not
tested

434445827

4345829

4345831

4345833

Not
tested

310

4323050

401114

401546,
402996

401546,
402996,
4313939

4312131

4318159

401546

Dye primer matrix standards.


We have tested this dye set but have not optimized for this instrument.

Installation standards
Installation standard

Source

For use with

DS-33 GeneScan Installation Standards

4376911

GeneScan 600 LIZ Size Standard v2.0


(6-FAM, VIC, NED, and PET dyes)

DS-33 GeneScan Installation Standards

4330397

GeneScan 500 LIZ Size Standard


(6-FAM, VIC, NED, and PET dyes)

DS-30 GeneScan Installation Standards

4316144

GeneScan 500 ROX


(6-FAM, HEX, and NED dyes)

DNA Fragment Analysis by Capillary Electrophoresis

197

Ordering Information
Reagent kits

Reagent kits
Item

Source

AFLP Kits
AFLP I Selective Primer Kit

4303050

AFLP

II Selective Primer Kit

4303051

AFLP

EcoRI Ligation/Amplification Module

402941

AFLP

MseI Ligation/Amplification Module

402942

AFLP

Amplification Core Mix Module

402005

SNaPshot Kits
SNaPshot Multiplex Kit

Contact your local


representative

SNaPshot Primer Focus Kit

4329538

GeneAmp EZ rTth RNA PCR Kit

N808-0178

GeneAmp

PCR Carryover Prevention Kit

N808-0068

AmpErase

UNG

N808-0096

For the Safety Data Sheet (SDS) of any chemical not distributed by Thermo Fisher Scientific, contact the chemical manufacturer. Before handling
any chemicals, refer to the SDS provided by the manufacturer, and observe all relevant precautions.

Other user-supplied materials


Item
Hi-Di Formamide, 25-mL

Source
4311320

Aerosol resistant pipette tips

MLS

Microcentrifuge tubes

MLS

Pipettors

MLS

Tape, labeling

MLS

Tube, 50-mL Falcon

MLS

Tube decapper, autoclavable

MLS

Deionized water, PCR grade

MLS

Vortex

MLS

Millipore

VS filter

CEPH Individual 1347-02 Control DNA

Millipore Part no. VSWP


02500
403062

For the Safety Data Sheet (SDS) of any chemical not distributed by Thermo Fisher Scientific, contact the chemical manufacturer. Before handling
any chemicals, refer to the SDS provided by the manufacturer, and observe all relevant precautions.

198

DNA Fragment Analysis by Capillary Electrophoresis

Documentation and Support

Thepublicationnumbersinthissectionareforthelatestproductversionsavailableat
thetimeofpublication.Fordocumentationfornewerproductversions,goto
www.lifetechnologies.com.

Instrument documentation
Publication
number

Document
Applied Biosystems 3500/3500xL Genetic Analyzer User Guide (3500 Series Software v1.0)
Applied

Biosystems

3500/3500xL Specification Sheet

Applied

Biosystems

3730 DNA Analyzer Getting Started Guide

Applied

Biosystems

3730/3730xl DNA Analyzer Specification Sheet

Applied

Biosystems

3130/3130xl Genetic Analyzers Getting Started Guide

4401661
106SP11-01

Applied Biosystems 3130/3130xl Specification Sheet

4359476
106SP08-01
4352715
106SP07-03

310 Genetic Analyzer User Guide

4317588

310 Genetic Analyzer Specification Sheet

903565

Veriti

4375799

Thermal Cycler User Guide

GeneAmp

PCR System 9700 User Guide

4303481

GeneMapper Software documentation


Publication
number

Document
GeneMapper Software Getting Started Guide: AFLP System Analysis

4403620

GeneMapper Software Getting Started Guide: Loss of Heterozygosity (LOH) Analysis

4403621

GeneMapper

4403672

Software Getting Started Guide: Microsatellite Analysis

GeneMapper

SNaPshot

Software Getting Started Guide:

GeneMapper

Software Reference and Troubleshooting Guide

4403614

GeneMapper

Software Installation and Administration Guide v4.1

4403614

GeneMapper

Software Reference and Troubleshooting Guide v4.1

4403673

GeneMapper Software Quick Reference Guide v4.1

DNA Fragment Analysis by Capillary Electrophoresis

Kit Analysis

4403618

4403615

199

Documentation and Support


Peak Scanner Software documentation

Peak Scanner Software documentation


Publication
number

Document
Peak Scanner Software Reference Guide

4382253

Peak Scanner Software Quick Reference Card

4383719

Application documentation
Publication
number

Document
AFLP applications
GeneMapper Software Getting Started Guide: AFLP System Analysis

4403620

AFLP Microbial Fingerprinting Protocol

402977

AFLP Plant Mapping Protocol

4303146

Aneuploidy applications
Aneuploidy Detection by QF-PCR of STR Markers on the Applied Biosystems 3500xL Genetic Analyzer

106AP28-01

BAC applications
BAC Fingerprinting on the Applied Biosystems 3730/3730xl DNA Analyzer

107AP04-01

Sizing of Large DNA Fragments Generated by BAC Fingerprinting on Capillary Electrophoresis System

106AP25-01

HiCEP applications
High coverage gene expression profiling on the Applied Biosystems 3500xL Genetic Analyzer

CO15884

ISSR applications
ISSR Genotyping of Endangered Plants Using an Optimized Workflow

106AP31-01

LOH applications
GeneMapper Software Getting Started Guide: Loss of Heterozygosity (LOH) Analysis
Relative Fluorescent Quantitation on CapillaryElectrophoresis Systems: Screening for Loss of Heterozygosity
in Tumor Samples on the Applied Biosystems 3130 Series Genetic Analyzers with GeneMapper
Software v3.7

4403621
106AP15-01

Methylation applications
Cells-to-CpG Bisulfite Conversion Kit (50) Protocol

4448998

Advances in Capillary Electrophoresis-Based Methods for DNA Methylation Analysis

106BR14-01

Three Optimized Workflows for CpG Island Methylation Profiling

106AP24-01

Microsatellite applications
GeneMapper Software Getting Started Guide: Microsatellite Analysis
Fact sheet: Microsatellite Analysis on the Applied

Biosystems

4403672

3130 Series Genetic Analyzers

Uniparental disomy (UPD) analysis of chromosome 15

106MI61-01
CO18249

Relative fluorescence quantitation applications


Aneuploidy Detection by QF-PCR of STR Markers on the Applied Biosystems 3500xL Genetic Analyzer

200

106AP28-01

DNA Fragment Analysis by Capillary Electrophoresis

Documentation and Support


Obtain SDSs

Publication
number

Document
Relative Fluorescent Quantitation on Capillary Electrophoresis Systems: Screening for Loss of Heterozygosity
in Tumor Samples on the Applied Biosystems 3130 Series Genetic Analyzers with GeneMapper Software
v3.7

106AP15-01

SNP applications
GeneMapper Software Getting Started Guide: SNaPshot Kit Analysis
the SNaPshot

User Bulletin: Using


Multiplex System with the POP-7 Polymer on Applied
3730/3730xl DNA Analyzers and 3130/3130

4403618
Biosystems

4367258

SNaPshot Multiplex Kit Quick Reference Card

4323975

Demonstration of SNP Genotyping Using a Single Nucleotide Extension Method and the 3500 Series Genetic
Analyzer

CO12863

Using the SNaPshot Multiplex System with the POP-7 Polymer on Applied Biosystems 3730/3730xl
DNA Analyzers and 3130/3130xl Genetic Analyzers

4367258

Obtain SDSs
SafetyDataSheets(SDSs)areavailablefromwww.lifetechnologies.com/support.
Note: FortheSDSsofchemicalsnotdistributedbyThermoFisherScientific,contact
thechemicalmanufacturer.

Obtain support
Forthelatestservicesandsupportinformationforalllocations,goto:
www.lifetechnologies.com/support
Atthewebsite,youcan:
AccessworldwidetelephoneandfaxnumberstocontactTechnicalSupportand
Salesfacilities
Searchthroughfrequentlyaskedquestions(FAQs)
SubmitaquestiondirectlytoTechnicalSupport
Searchforuserdocuments,SDSs,vectormapsandsequences,applicationnotes,
formulations,handbooks,certificatesofanalysis,citations,andotherproduct
supportdocuments
Obtaininformationaboutcustomertraining
Downloadsoftwareupdatesandpatches

DNA Fragment Analysis by Capillary Electrophoresis

201

Documentation and Support


Obtain support

202

DNA Fragment Analysis by Capillary Electrophoresis

References

Ammiraju,J.S.S.,Dholakia,B.B.,Santra,D.K.etal.2001.Identificationofintersimple
sequencerepeat(ISSR)markersassociatedwithseedsizeinwheat.TheorApplGenet.
102:726732.
Andersen,P.S.,Jespersgaard,C.,Vuust,J.etal.2003.Capillaryelectrophoresisbased
singlestrandDNAconformationanalysisinhighthroughputmutationscreening.
HumMutat.21:455465.
Anderson,G.R.,Brenner,B.M.,Swede,H.etal.2001.Intrachromosomalgenomic
instabilityinhumansporadiccolorectalcancermeasuredbygenomewide
allelotypingandinter(simplesequencerepeat)PCR.CancerRes.61:82748283.
Blair,M.W.,Panaud,O.,McCouch,S.R.1999.Intersimplesequencerepeat(ISSR)
amplificationforanalysisofmicrosatellitemotiffrequencyandfingerprintinginrice
(OryzasativaL.).TheorApplGenet.98:780792.
Canzian,F.Salovaara,R.,Hemminki,A.etal.1996.SemiautomatedAssessmentofLoss
ofHeterozygosityandReplicationErrorinTumors.CancerRes.56:33313337.
Davis,L.M.,Glenn,T.C.,Strickland,D.C.etal.2002.MicrosatelliteDNAanalyses
supportaneastwestphylogeographicsplitofAmericanalligatorpopulations.JExp
Zool.294:35272.
Derakshani,M.,Lukow,T.andLiesack,W.2001.Novelbacteriallineagesatthe(sub)
divisionlevelasdetectedsignaturenucleotidetargetedrecoveryof16srRNAgenes
frombulksoilandricerootsoffloodedricemicrocosms.AppliedEnvironmental
Microbiology.67(2):623631.
Doyle,J.J.,DoyleJ.L.1987.ArapidDNAisolationprocedureforsmallquantitiesof
freshleaftissue.PhytochemBull.19:1115.
Ellegren,H.2004.Microsatellites:simplesequenceswithcomplexevolution.NatRev
Genet.5:43545.
Eschenhagen,M.,Schuppler,M.,andRoske,I.2003.Molecularcharacterizationofthe
microbialcommunitystructureintwoactivatedsludgesystemsfortheadvanced
treatmentofdomesticeffluents.WaterRes.37(13):32243232.
Fleischer,R.C.andLowe,S.1995.Constructionandscreeningofmicrosatellite
enrichedgenomiclibraries.InFerraris,J.andPalumbi,S.(eds),MolecularZoology:
Advances,StrategiesandProtocols.WileyLiss,NY,459468.
Goel,S.,Chen,Z.,Akiyama,Y.,Connor,J.A.,etal.2006.Comparativephysical
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squamulatumandCenchrusciliaris.Genetics.173(1):389400.
Gupta,S.K.,Souframanien,J.,andGopalakrishna,T.2008.Constructionofagenetic
linkagemapofblackgram,Vignamungo(L.)Hepper,basedonmolecularmarkersand
comparativestudies.Genome.51:628637.

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Hauge,X.Y.,andLitt,M.1993.Astudyoftheoriginofshadowbandsseenwhen
typingdinucleotiderepeatpolymorphismsbythePCR.HumMolGenet.2:411415.
Holleley,C.E.,andGeerts,P.G.,2009.MultiplexManager1.0:acrossplatform
computerprogramthatplansandoptimizesmultiplexPCR.BioTechniques46:511517.
Huelsenbeck,J.P.,andRonquist,F.2001.MRBAYES:Bayesianinferenceofphylogeny.
Bioinformatics17:754755.
Inazuka,M.,Tahira,T.,andHayashi,K.1996.OnetubepostPCRfluorescentlabeling
ofDNAfragments.GenomeRes.6:551557.
Inazuka,M.,Wenz,H.M.,Sakabe,M.,Tahira,T.,andHayashi,K.1997.Astreamlined
mutationdetectionsystem:multicolorpostPCRfluorescencelabelingand
singlestrandconformationalpolymorphismanalysisbycapillaryelectrophoresis.
GenomeRes.7:10941103.
Iwahana,H.,Adzuma,K.,Takahashi,Y.,Katashima,R.,Yoshimoto,K.,andItakura,M.
1995.MultiplefluorescencebasedPCRSSCPanalysiswithpostlabeling.GenomeRes.
4:275282.
Johnson,E.L.,Zhang,D.,Emche,S.D.etal.1994.Constructionoflibrariesenrichedfor
sequencerepeatsandjumpingclones,andhybridizationselectionforregionspecific
markers.ProcNatl.Acad.Sci.USA91:8892.
Lai,Y.andSun,F.,Therelationshipbetweenmicrosatelliteslippagemutationrateand
thenumberofrepeatunits.2003.MolecularBiologyandEvolution.20(12):21232131.
Leroy,X.J.,andLeon,K.2000.Arapidmethodfordetectionofplantgenomic
instabilityusingunanchoredmicrosatelliteprimers.PlantMolBiolRep.2000.
18:283a283g.
Luo,M.C.etal.,Highthroughputfingerprintingofbacterialartificialchromosomes
usingtheSNaPshotlabelingkitandsizingofrestrictionfragmentsbycapillary
electrophoresis,Genomics82(2003)378389.
MolecularCloning.1987.SecondEdition.ColdSpringHarborLaboratory,NY:Cold
SpringHarborLaboratoryPress.
Murray,V.,Monachawin,C.,andEngland,P.R.1993.Thedeterminationofthe
sequencespresentintheshadowbandsofadinucleotiderepeatPCR.NucleicAcids
Research.21:23952398.
Orita,M.,Iwahana,H.,Kanazawa,H.etal.1989.InterandIntraspecificVariation
amongFiveErythroxylumTaxaAssessedbyAFLP.ProcNatlAcadSciUSA
86:27662770.
Osborn,A.M.,Moore,R.B.,andTimmis,K.N.2000.Anevaluationofterminal
restrictionfragmentlengthpolymorphism(TRFLP)analysisforthestudyofmicrobial
communitystructureanddynamics.EnvironmentalMicrobiology.2(1):3950.
Naimuddin,M.andNishigaki,K.2004.Genomeanalysistechnologies:Towards
speciesidentificationbygenotype.BriefingsinFunctionalGenomicsandProteomics
1(4):356371.
Ronquist,F.,andHuelsenbeck,J.P.2003.MRBAYES3:Bayesianphylogeneticinference
undermixedmodels.Bioinformatics19:15721574.

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Sakamoto,M.,Takeuchi,Y.,Umeda,M.,Ishikawa,I.andBenno,Y.2003.Applicationof
terminalRFLPanalysistocharacterizeoralbacterialflorainsalivaofhealthysubjects
andpatientswithperiodontitis.JMedMicrobiol.52:7989.
Savelkoul,P.H.M.,Aarts,H.J.M.,deHaas,J.etal.1999.Amplifiedfragmentlength
polymorphismanalysis:thestateofanart.JClinMicrobiol.37(10)30833091.
Serra,R.,Cabanes,F.J.,Perrone,G.,Venancio,A.,Mule,G.,andKozakiewicz,Z.2006.
Aspergillusibericus:anewspeciesofsectionNigriisolatedfromgrapes.Mycologia
98(2)295306.
Shihabi,Z.K.andHinsdale,M.E.1995.Somevariablesaffectingreproducibilityin
capillaryelectrophoresis.Electrophoresis.16:21592163.
Shinde,D.,Lai,Y.,Sun,F.,andArnheim,N.2003.TaqDNApolymeraseslippage
mutationratesmeasuredbyPCRandquasilikelihoodanalysis:(GA/GT)nand(A/T)n
microsatellites.NucleicAcidsRes.31:974980.
Topal,M.D.,andFresco,J.R.1976.Complementarybasepairingandtheoriginof
substitutionmutations.Nature263:285289.
UBCPrimerSet9,2006.www.michaelsmith.ubc.ca/services/NAPS/Primer_Sets/
Primers_Oct2006.pdf
WanQH.,Wu,H.,Fujihara,T.,andFang,SG.2004.Whichgeneticmarkerforwhich
conservationgeneticsissue?Electrophoresis.25:21652176.
Wang,H.Z.,Wu,Z.X.,Lu,J.J.etal.2009.Moleculardiversityandrelationshipsamong
Cymbidiumgoeringiicultivarsbasedonintersimplesequencerepeat(ISSR)markers.
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Witmer,P.D.,Doheny,K.F.,Adams,M.K.,Boehm,C.D.etal.,2003.Thedevelopmentof
ahighlyinformativemousesimplesequencelengthpolymorphism(SSLP)markerset
andconstructionofamousefamilyfreeusingparsimonyanalysis.GenomeRes.
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Wolfe,A.D.,Xiang,QY.,andKephart,S.R.1998.Assessinghybridizationinnatural
populationsofPenstemon(Scrophulariaceae)usinghypervariableintersimplesequence
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Zhao,H.,Bughrara,S.S.,andOliveira,J.A.2006.Geneticdiversityincolonialbentgrass
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49(4):328335.

DNA Fragment Analysis by Capillary Electrophoresis

205

References

206

DNA Fragment Analysis by Capillary Electrophoresis

Glossary

allele

Onespecificsequenceofalocus.Differentallelesofasinglelocuswillhaveslightly
differentDNAsequences.

amplicon

TheproductofaPCRreaction.Typically,anampliconisashortpieceofDNA.

association studies

Studiesthatinterrogateadensesetofmarkerstoidentifyassociationsbetweenthose
markersandthelociofinterest.Associationstudiesaremorepowerfulthanlinkage
mappingstudiesforthedetectionofweaksusceptibilityloci.

backcross

AgeneticcrosswhereanindividualfromtheF1generationismatedtoanindividual
withthegenotypeofoneoftheparents.

bin

InGeneMapperSoftware,theexpectedlocationofanindividualpeakorallele,
definedbyabasepairrangeandacolor.Youtypicallydefineabinforeachpossible
alleleassociatedwithamarker.

binset

InGeneMapperSoftware,asetofbinsthatyouspecifyintheanalysismethod.

contig

InBACfingerprinting,acontiguoussequenceofDNAcreatedbyassembling
overlappingsequencedfragmentsofachromosome.Agroupofclonesrepresenting
overlappingregionsofthegenome.

diploid

Havingtwocopiesofeverychromosome.

dye set

Thetermdyesetcorrespondsto:
Thephysicaldyesetusedforlabelingfragments.
Thesoftwareselectionyoumakethatidentifiesthedyecolorsinthedyeset,the
orderofthedyepeaksinthedyeset,andspectralanalysisparametersforthe
dyes.

electrophoresis

Theactofapplyinganelectricalfieldtoaporoussubstrateinordertoseparate
moleculesbysizeorshape.Forexample,nucleicacidslikeDNAandRNAare
negativelychargedandarethusattractedtoapositivechargeandwillmovetowardit
throughamatrixlikeapolymeroragarosegel.Thestrengthoftheelectricalfieldand
chargeonthemoleculesandthesizeoftheporesinthematrixdeterminethespeedat
whichdifferentmoleculesinthestartingmixturemigrate.Ingeneral,smaller
moleculesmigratefasterthanlargermoleculesbecausetheyarelessobstructedasthey
travelthroughthematrix.

emission spectrum

Theintensityofemittedlight(fluorescence)asafunctionofthewavelengthofthe
emittedlight.

DNA Fragment Analysis by Capillary Electrophoresis

207

Glossary

excitation efficiency

Theprobabilitythatitwillabsorblightofacertainwavelength,asapercentageofthe
probabilityofabsorptionatthewavelengthofmaximumabsorption.

excitation spectrum

Theintensityofemittedlightasafunctionofthewavelengthoftheexcitinglight.

filter set

Physicalfiltersthatseparatedyesignalsinoldergelbasedcapillaryelectrophoresis
instruments.NewerThermoFisherScientificinstrumentsuseadiffractiongrating.

fingerprint

Acharacteristicpatternofpeaksorbandsafteranamplificationordigestofgenomic
DNAthatcanbeusedtoidentifyanindividual.

genetic mapping

Analysisoftheprogenyfromgeneticcrossestodeterminetherelativepositionof
chromosomallocations.

Linkage mapping

Ameioticmappingtechniquewhichsearchesforlinkagebetweenamarkerandthe
diseasegenewithinseveralgenerationsofaffectedfamilies.Thesestudiesexaminethe
inheritanceofmarkersinbothaffectedandunaffectedmembersofthefamilyandthen
determinewhetherparticularmarkersarephysicallyclosetothediseasegeneof
interest.Linkagemappingcanbeusedtoscantheentiregenomerelativelyeasily.

ISSR PCR

IntersimplesequencerepeatPCR.SeeIntersimplesequencerepeat(ISSR)PCRon
page137.

locus

Alocationonachromosome.Thetermissometimesusedmorenarrowlytodescribe
thelocationofageneorgeneticmarker.(Plural:loci).SeealsoMarker.

marker

Anyobservablegeneticcharacteristic(forexample,gene,phenotype,microsatellite
sequence,SNP)thatcanbeusedtoidentifyageneticlocation.Tobeusefulingenetic
studies,amarkermustbepresentindifferentformstoallowresearcherstodistinguish
betweenindividuals.Onemarkerrepresentsonelocusandoneprimerpair.Defined
bysizerangeofexpectedallelesanddye(color)attachedtoprimer.

microsatellites

Highlyrepetitivesimplesequencerepeatsof2to7basepairs,alsocalledshorttandem
repeats(STRs).Microsatellitesfallintothebroadercategoryreferredtoasvariable
numberoftandemrepeats(VNTRs),whichalsoincludesminisatellites.

minisatellites

Highlyrepetitivesimplesequencerepeatsrangingfromapproximately10to30base
pairs.Minisatellitesfallintothebroadercategoryreferredtoasvariablenumberof
tandemrepeats(VNTRs),whichalsoincludesmicrosatellites.

panel

InGeneMapperSoftware,acollectionofexpectedsizerangesanddyecolors
(markers)associatedwitheachprimerpair.

phylogenetic studies

Studiestodetermineevolutionaryrelationshipsbetweenorganisms(forexample,
speciesorpopulations).

polymerase

Anenzymethatcatalyzespolymerization.DNAandRNApolymerasesbuild
singlestrandedDNAorRNA(respectively)fromfreenucleotides,usinganother
singlestrandedDNAorRNAasthetemplate.

polymorphic locus

Alocuswithmorethanoneallelethatiscommonlyfoundinapopulation.

208

DNA Fragment Analysis by Capillary Electrophoresis

Glossary

population study

Astudythattypesgeneticmarkersonalargepopulationtoidentifyassociations
betweenamarkerandaphenotype.

primer

AshortsinglestrandofDNAthatservesastheprimingsiteforDNApolymeraseina
PCRreaction.

pull-up peaks

Artifactpeaksinoneormoredyecolorsthatarecausedbyhighorsaturatedsignal
intensityinanotherdyecolor.

quantum yield

Theprobabilitythatadyeinitsexcitedstatewillemitaphotonasitdecaysbacktothe
groundstate.

restriction enzymes

EndonucleasesthatcleavethephosphatebackboneofdoublestrandedDNAathighly
specificsequencescalledrestrictionsites.

template

InPCR,thenucleicacidmoleculethatprovidesthesequencetoamplify.Forexample,
genomicDNAcanbethetemplateforaPCRreactionthatamplifiesaspecificregion
withinthegenome.

Variable Number
Tandem Repeat
(VNTR)

Anygeneswhoseallelescontaindifferentnumbersoftandemlyrepeated
oligonucleotidesequences.

DNA Fragment Analysis by Capillary Electrophoresis

209

Glossary

210

DNA Fragment Analysis by Capillary Electrophoresis

Index

Numerics

2720ThermalCycler 58
3Anucleotideaddition 33
310instrument
Seealsocapillaryelectrophoresis
dyesetsandmatrixstandards 86
matrix,overview 87
runmodule,updatingforGeneScanLIZSize
Standard 46
safetyinformation 68
signalintensityrange 77, 158
specifications 68
3130Seriesinstrument
Seealsocapillaryelectrophoresis
dyesetsandmatrixstandards 86
runmodule,updatingforGeneScanLIZSize
Standard 48
runmodules,downloading 48
safetyinformation 68
signalintensityrange 77, 158
specifications 68
3500Seriesinstrument
Seealsocapillaryelectrophoresis
dyesetsandmatrixstandards 86
performance 70
runmodules 69
safetyinformation 68
signalintensityrange 77, 158
specifications 68
3730Seriesinstrument
Seealsocapillaryelectrophoresis
dyesetsandmatrixstandards 86
runmodule,downloading 48
runmodule,optimizingforGeneScan600LIZSize
Standard 48
runmodules 69
safetyinformation 68
signalintensityrange 77, 158
specifications 68
throughput 69

AccuPrimeTaqDNAPolymerase 22, 24
AccuPrimeTaqDNAPolymeraseHighFidelity 22
AFLP
advantages 126
analysismethod,GeneMapperSoftware 129
applications 127
dataanalysis 129
exampledata 129
experimentandprimerdesign
recommendations 127
genotypesinGeneMapperSoftware 130
instrumentandconsumable
recommendations 127
overview 125
PCRkits 198
principle 126
restrictionenzymes 128
workflow 128
agarosegel,usingfortroubleshooting 157
ambienttemperature,problemscausedby 160
amplification
SeealsoPCR
nonspecific 32
postamplificationmanipulations 33
selective 31
amplifiedfragmentlengthpolymorphism.SeeAFLP
AmpliTaqDNAPolymerase 22, 24
AmpliTaqDNAPolymerase,LD 22, 24
AmpliTaqGoldDNAPolymerase 23, 24
analysissoftware 89
aneuploidy,relativefluorescenceapplication 17
animalbreeding,microsatelliteapplication 16
animaltyping,microsatelliteapplication 16
annealingtemperature
optimizing 64
PCRparametersforunknown 61, 63
annealing,factorsaffecting 30
associationstudies 110
autosampler,problemscausedbymisalignment 161

DNA Fragment Analysis by Capillary Electrophoresis

211

Index

B
BACfingerprinting
applications 133
dataanalysis 73, 90, 159
experimentandprimerdesign
recommendations 134
instrumentandconsumable
recommendations 134
overview 132
principle 133
workflow 135
bacterialartificialchromosome.SeeBACfingerprint
ing
baselinetroubleshooting 178
basepairingenergies 30, 31
bleedthroughpeaks.Seepulluppeaks
breeding 110

C
cancerprogressionanalysis 110
capillaryarray
foreachinstrument 68
problemscausedbydegradedorclogged 160
capillaryelectrophoresis
Seealso310instrument; 3130Series
instrument; 3500Seriesinstrument; 3730
Seriesinstrument
controlDNA 57
definition 18
factorsaffecting 82
injectiontime,optimizing 79
injectionvoltage,optimizing 80
optimizing 67
optimizingconditions 80
polymers 83
runmodules 46
runtime,optimizing 81
runvoltage,optimizing 81
signalintensity 77
spatialcalibration 84
spectralcalibration 84
troubleshooting 180
CE.Seecapillaryelectrophoresis
CEPH134702ControlDNA 57, 157
CNV,relativefluorescenceapplication 17
concentration
DNAtemplate 59
dNTP 59

212

enzyme 60
Mgion 59
sample 158
controls
CEPH134702ControlDNA 157
controlDNA,using 57
DNAtemplate 157
process 157
recommendationforfrequency 69, 156
runningtoisolateaproblem 156
CopyNumberVariation.SeeCNV
customprimerdesign 29

D
data
checkingquality 104, 153
electropherogramqualityrequirements 104
troubleshooting 153
dataanalysis
GeneMapperSoftware 89
PeakScannerSoftware 92
desalting 190
dinucleotiderepeats 115
DNAmethylation 149
DNApolymeraseenzymes
3Aaddition 33
characteristics 24
recommended 22
DNAtemplate
concentration 59
control 157
isolating 55
purifying 56
quantifying 56
storing 56
dyesets
dyecomponents 41
matrixstandardsfor 86
dyes
chemicalforms 36
emission 38
emissionmax 38

E
electropherogram,quality 104
electrophoresis.Seecapillaryelectrophoresis
EPTtrace

DNA Fragment Analysis by Capillary Electrophoresis

Index

examining 155
exampleofgoodquality 155
troubleshooting 180
errormessage,GeneMapperSoftware 154
evaluatingdata
electropherogram 104
stutter 113
excitationandemissionmax 38
experimentaldesignconsiderations 21

F
fingerprinting
AFLP 125
applications 17
BAC 132
HiCEP 136
ISSR 137
TRFLP 131
fluorescentlabeling 25
forensics.SeeHumanIdentification
fragmentanalysis
applications 16
definition 15
workflow 19

G
GeneAmpGoldFastPCRMasterMix 23
GeneAmpPCRSystem9700,384WellDual
Autolid 58
GeneAmpPCRSystem9700,Dual384well 58
GeneAmpPCRSystem9700,Dual96Well 58
GeneMapperSoftware
AFLPdefaultmethod 129
AFLPgenotypes 130
autoanalysis 91
errormessage 154
features 91
ISSRbins 139
microsatellitedefaultmethod 112
peakdetectionsettings 94
peakstartandendsettings 97
sizing 98
sizingmethods 100
SNaPshotdefaultmethod 123
troubleshooting 187
workflow 93
generalPCR,thermalcyclerparameters 61, 62

DNA Fragment Analysis by Capillary Electrophoresis

GeneScansizestandards.Seesizestandard
geneticanalyzer.See310instrument; 3130Series
instrument; 3500Seriesinstrument; 3730Se
riesinstrument
geneticdiversity
fingerprintingapplication 17
microsatelliteapplication 16
geneticmapsfornewspecies,fingerprinting
application 17
genomescans 110
guidelines
PCRcontamination 64
PCRparameters 63
primerdesign 29
sizecalling 90

H
hairpinsecondarystructures 31
HiCEP 136
applications 136
overview 136
principle 136
workflow 136
HID.SeeHumanIdentification
HiDiformamide
issuescausedbyimproperlystored 159
storageconditions 82
highcoverageexpressionprofiling.SeeHiCEP
hotstartPCR
description 60
enzyme 23
thermalcyclerparameters 60, 62
HumanIdentificationapplications.Gotolifetechnolo
gies.com
humidity,problemscausedby 161

I
injection
factorsaffecting 78
time,optimizing 79
voltage,optimizing 80
instruments
forusewithguide 13
platetypes 58
problemscausedby 160
RFUranges 158
runmodules 46
signalintensityranges 158

213

Index

troubleshooting 158, 159, 180


Intersimplesequencerepeat.SeeISSR
ionicstrengthofbuffer 78
ISSR
applications 138
creatingmultiplebins 139
dataanalysis 139
exampledata 140
experimentandprimerdesign
recommendations 139
microsatelliteapplication 16
overview 137
principle 137
workflow 139

L
labeling,fluorescent 25
laboratorywater,problemscausedby
contamination 159
linkagegroupsamongcrosses,fingerprinting
application 17
linkagemapping
fragmentanalysis 16
microsatelliteanalysis 110
thermalcyclerparameters 62
LOH
microsatelliteapplication 16
relativefluorescenceapplications 17
LossofHeterozygosity.SeeLOH
lowcopyamplification,enzyme 22, 23

M
matrix
310instrument 191
standardsforspectralcalibration 86
methylationassessment 149
microbialgenemapping,fingerprinting
application 17
microsatelliteanalysis
advantages 108
applications 110
dataanalysis 112
experimentandprimerdesign
recommendations 111
instrumentandconsumable
recommendations 111
overview 107
principle 108

214

sizestandard,GeneScan400HDROX 49
sizestandard,GeneScan600LIZ 111
stutter 113
troubleshooting 113
workflow 112
microsatelliteinstability,microsatellite
application 16, 146
migrationtroubleshooting 168
MLPA,relativefluorescenceapplications 17, 143
MLVA,microsatelliteapplication 16
multicomponentanalysis 26, 37
MultilocusVariantAnalysis.SeeMLVA
multiplexing
benefitsandlimitations 26
guidelines 28
poolingratios 27
software 28
strategies 27
troubleshooting 29

N
noise,troubleshooting 178
normalizationsizestandard 45

P
parentageanalysis 110
paternitytesting 110
pathogensubtyping,microsatelliteapplication 16
PCR
Seealsothermalcyclerparameters
3addition 33
carryover 65
contamination 64
controlDNA 57
DNAtemplate 55
general 61, 62
hotstart 60, 62
linkagemapping 62
maximizeyieldofspecificproducts 61, 63
nonspecificamplification 32
plates 58
postamplificationmanipulations 33
primerdesign 29
primers,preparing 57
problemscausedbyexpiredreagents 159
products,storing 56
reactionvolume 58

DNA Fragment Analysis by Capillary Electrophoresis

Index

reagentconcentrations 59
selectiveamplification 31
setup 64
sidereactions 60
templatevolume 58
timerelease 61, 62
touchdown 61, 63
troubleshooting 176
XL 63
PCRworkareas 64
peakdetection
peakwindowsize 94
polynomialdegree 94
peakmorphologytroubleshooting 169
PeakScannerSoftware
features 92
overview 92
phylogeneticstudies 110
plantgenomemapping,fingerprintingapplication 17
planttyping,microsatelliteapplication 16
PlatinumMultiplexPCRMasterMix 23, 24
PlatinumPfxDNAPolymerase 23, 24
polymer
characteristics 83
handling 83
problemscausedbydegradedorexpired 159
types 83
polymeraseenzymes.SeeDNApolymeraseenzymes
polynomialdegree
peakdetection 94
varying 95
windowsizevalue 96
poolingratiosformultiplexing 27
POPpolymer.Seepolymer
populationgeneticsstudies 110
postamplificationmanipulations 33
preamplificationtroubleshooting 190
primer/dimer
minimizing 62
primer/dimer,minimizing 60
primers
designguidelines 29
reconstituting 57
SNaPshot 123
tailing 34

DNA Fragment Analysis by Capillary Electrophoresis

Q
QFPCR,relativefluorescenceapplications 17, 143
QualitativeFluorescencePCR.SeeQFPCR
quantification
DNAtemplate 56
primers 57
QuantitativeMultiplexPCRofShortFluorescentFrag
ments.SeeQMPSF

R
rawdata
examine 154
example 155
reagenttroubleshooting 158, 159
RelativeFluorescenceQuantitation.SeeRFQ
Replicationerror 146
RER 146
resolution
definitionof 79
formula 79
troubleshooting 169
RFQ
applications 17, 144
dataanalysis 145
determiningrelativenumberofmolecules 146
determiningrelativequantities 145
experimentandprimerdesign
recommendations 144
LOHworkflow 145
minimizingsignalintensityvariation 144
overview 143
peakheightversusarea 143
RFUrangesforinstruments 158
rTthDNApolymerase
description 24
thermalcyclerparameters 63
XL 24
runmodules 46

S
SafetyDataSheets(SDSs),obtaining 201
safetyinformation 55
saltconcentration
desalting 190
impactonelectrophoresis 82
sample
concentration,problemscausedbyhigh 158

215

Index

contamination,problemscausedby 158
samplesaltconcentration.Seesaltconcentration
secondarystructure
primer 30
template 31
selectiveamplification 31
signalintensity
minimizingvariation 144
optimizing 77
rangeforeachinstrument 77, 158
relativeorderofdyes 38
troubleshooting 164
SingleNucleotidePolymorphism.SeeSNP
singleplexing 26
SizeMatchEditor,displaying 153
sizemethods,GeneMapperSoftware 100
sizestandards
function 42
GS1000ROX 51
GS120LIZ 44
GS1200LIZ 47
GS350ROX 49
GS400HDROX 49
GS500LIZ 44
GS500ROX 50
GS600LIZ 45
GS600LIZv2.0 45
modifyingdefinition 153
normalization 45
preparation 43
storage 43
troubleshooting 182
sizecallingguidelines 90
sizing
CubicSplinemethod 101
curve 100
GlobalSouthernmethod 103
howtheGeneMapperSoftwareperforms 98
LeastSquaresmethod 100
LocalSouthernmethod 102
methods 100
troubleshooting 153, 182
sizingquality
checking 153
troubleshooting 182
software,analysis 89
spatialcalibration,overview 84
spectralcalibration

216

matrixstandards 41
overview 84
splitpeaks,incomplete3Aaddition 33
SQ.Seesizingquality
standards
Seealsosizestandards 42
installstandardfortroubleshooting 157
matrixforspectralcalibration 86
stutter
identifying 113
troubleshooting 113
SuperScriptIIIReverseTranscriptase 23, 24
support,obtaining 201

T
tailingprimer 34
technicalsupport 201
template
DNA 56, 59
smallvolume 58
terminalrestrictionfragmentlengthpolymorphism.
SeeTRFLP
thermalcyclerparameters
generalPCR 61, 62
hotstart 60, 62
linkagemapping 62
optimizing 63
timereleasePCR 61, 62
touchdownPCR 61, 63
XLPCR 63
thermalcyclers
applications 58
safetyinformation 55
specifications 58
timereleasePCR
enzyme 23
thermalcyclerparameters 61, 62
Tm
calculator 29
factorsaffecting 30
primer 29
touchdownPCR,thermalcyclerparameters 61, 63
training,informationon 201
TRFLP
applications 131
dataanalysis 132
experimentandprimerdesign
recommendations 132
DNA Fragment Analysis by Capillary Electrophoresis

Index

instrumentandconsumable
recommendations 131
overview 131
principle 131
troubleshooting
agarosegel,running 157
ambienttemperature 160
autosampleralignment 161
baseline 178
bufferstrength 160
capillaryarray 160
capillaryelectrophoresis 180
CEPH134702ControlDNA 157
consumableissues 159
dataquality 153
EPTtrace 180
errormessage 187
extrapeaks 172
GeneMapperSoftware 187
instrumentissues 160, 180
isolatingtheproblem,controls 156
matrix 161
microsatelliteanalysis 113
migration 168
multiplexing 29
noise 178
PCR 176
peakmorphology 169
preamplificationgel 190
processcontrol 157
reagentissues 159
resolution 169
sampleissues 158
signalintensity 164
sizestandard 182
sizingandsizingquality 182
stutter 113
temperatureandhumidity 161
workflow 152
TthDNApolymerase 24

amplifiedDNA 65
PCRsetup 64

X
XLPCR,thermalcyclerparameters 63

V
Veriti384WellThermalCycler 58
Veriti96WellThermalCycler 58
VNTR 109

W
workarea
DNA Fragment Analysis by Capillary Electrophoresis

217

Index

218

DNA Fragment Analysis by Capillary Electrophoresis

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05 April 2014