Kt99 Single Nucleotide Polymorphism SNPTK

Single Nucleotide Polymorphism (SNP) GeNei™ Single Nucleotide Polymorphism (SNP) GeNei™
GeNei™ Single Nucleotide

Polymorphism (SNP)
Demonstration
Teaching Kit Manual
Cat No.: 6109900011730

Revision No.: 00050516
 Genei Laboratories Private Limited, 2016  Genei Laboratories Private Limited, 2016
CONTENTS
 Objective 1
 Principle 1
 Kit Description 4
 Materials Provided 5
 Procedure 6
 Observation & Interpretation 8
Agarose Gel Electrophoresis
 Introduction 11
 Principle 11
 Procedure` 14
 Ordering Information 16
19
Objectives:
 To detect single nucleotide polymorphism using SNP
specific primers by PCR
 To Establish genetic variation (on the basis of SNPs)
Principle:
Single Nucleotide Polymorphism or SNPs (pronounced
“snips”) are DNA sequence variations /changes that can occur
within an individuals DNA sequence. The Genetic code is
specified by the four nucleotides A (Adenine),
G (Guanine), C (Cytosine) and T (Thymine). SNP occurs
when a single nucleotide (A, T, G or C) in the DNA sequence
is altered.
An example for SNP is AAGGTTA is altered to ATGGTTA

where the second nucleotide (A) in the first snippet is
replaced by T in the second snippet.
PCR based SNP detection facilitates scientific research in

variety of fields ranging from population genetics and
evolutionary biology to large-scale disease and drug-
associated studies.
In this technique selected regions of a DNA sequence from

multiple individuals sharing a common trait are compared.
1 18
The PCR includes 3 primers, one specific for the SNP region
and one specific for a consensus region. One of the primers
(forward or reverse) is common to both normal and SNP type
DNA. The consensus primers will amplify the consensus
region (an 800 bp fragment) in both normal and SNP type
DNA, while the SNP specific primers will amplify a 550 bp
fragment from the SNP type DNA and not normal DNA (refer
fig. 1 and fig. 2). This is because the SNP specific primer will
bind to normal DNA inadequately forming mismatch at the 3’-
end where as in the SNP type DNA it makes a perfect match.
Note: This kit contains no genomic DNA and the primers
are not specific to the human genome.
No. amplification seen

due to mismatch Primer 3 Primer 2
Primer 1
Fig 1: Normal DNA showing 800 bp fragment amplification
17 2
Ordering Information
550 bp amplification seen Product Size Cat #
GeNei™ Single Nucleotide 1 EA 6109900011730

Primer 3 Primer 2 Polymorphism (SNP) Demonstration
Teaching Kit
Primer 1 (Consumables for 5 Experiments)
Email:
800 bp DNA amplification seen Sales: sales@geneilabs.com
Fig 2: SNP type DNA showing amplification of 550 bp and 800 Customer Support: techsupport@geneilabs.com
bp fragments
Primer 1: Common Forward Primer

Primer 2: Reverse Primer (Specific for consensus region)
Primer 3: SNP Primer (Specific for detection of SNP)
16
3
Electrophoresis Kit Description:
7. Pour 1X TAE buffer into the gel tank till the buffer level Two different DNA samples (normal and SNP type) are
stands at 0.5 to 0.8 cm above the gel surface. provided along with a primer mix containing 3 different
8. Gently lift the combs, ensuring that wells remain intact. primers required for PCR. Primers specific to the SNP will
9. Connect the power cord to the electrophoretic power amplify only if the SNP is present but the consensus primers
supply according to the convention will amplify both in the normal as well as the SNP type DNA.
red: anode, black: cathode. Thus , the PCR product from the SNP type DNA will show 2
10. Load the samples in the wells in the desired order. bands (800bp and 550bp) whereas the PCR product from the
11. Set the voltage to 50 V and switch on the power normal DNA will show only one band (800 bp Refer Fig 3).
supply. Students will carry out PCR using the 2 templates and the
12. Switch off the power when the tracking dye primer mix, analyze the PCR products on agarose gel and
th
(bromophenol blue) from the well reaches ¾ of the determine which DNA has the SNP.
gel. This takes approximately one hour.
616109900011730 : The kit is designed to demonstrate
Staining Procedure to Visualize DNA SNP detection where two DNA
13. Prepare 1X staining dye by diluting 6X dye (1:6) templates, one normal and the other
(Cat No. 612601480061730) with distilled water. template with SNP are used for PCR
(Approximately 50 ml of 1X Staining Dye is required for amplification. The kit provides
one experiment. Therefore, make up 8 ml of 6X dye to enough reagents for conducting
48 ml with distilled water). 5 experiments.
14. Carefully transfer the gel (from gel tank) into a tray
containing 1X staining solution. Make sure that the gel Duration of the Experiment: Approximately 5 hours.
is completely immersed.
15. For uniform staining, place the tray on a rocker for
approximately one hour or shake intermittently every
10 to 15 minutes.
16. Pour out the staining dye into a container. (The dye
can be reused twice). Destain the gel by washing with
tap water several times till the DNA is visible as a dark
band against a light blue background.
4
15
Materials Provided: Procedure:
The list below provides information about the materials
Preparation of 2% Agarose Gel
supplied in the kit. The components should be stored as
suggested. 1. Prepare 1X TAE by diluting appropriate amount of 50X
TAE buffer. For one experiment, approximately 200 ml
Quantity Store of 1X TAE is required. (Dilute 4 ml of 50X TAE to 200
ml with distilled water).
Materials 6109900011730
2. Weigh 1 g of agarose and add to 50 ml of 1X TAE.
(5 Expts.) This gives 2% agarose.
°
Template DNA 1 5 µl -20 C
3. Boil till agarose dissolves completely and a clear
Template DNA 2 5 µl -20°C solution is obtained.
°
Primer Mix 40 µl -20 C 4. Add 2 µl Ethidium bromide to molten agarose (from a
° stock of 10 mg/ml in water), when temperature is
10X Assay Buffer 50 µl -20 C
10 mM dNTP Mix 20 µl
°
-20 C around 50°C. Mix and cast the gel.
StepUp™ 100 bp DNA 50 µl -20°C 5. Meanwhile place the comb of electrophoresis set such
Ladder (Ready to use) that it is approximately 2 cm away from the cathode.
Taq DNA Polymerase 5 µl -20°C 6. Pour the agarose solution in the central part of tank
(3 U/µl) when the temperature reaches approximately 60ºC.
0.25 ml -20°C Do not generate air bubbles. The thickness of the gel
2.5X Gel Loading Buffer
° should be around 0.5 to 0.9 cm. Keep the gel
Nuclease Free Water 1 ml -20 C undisturbed at room temperature for the agarose to
Mineral Oil 0.25 ml RT solidify.
Agarose 5g RT
50X TAE 20 ml RT
PCR vials 10 Nos RT
Note: Rooom Temperature (RT) not exceed 20°C to 25°C.
Materials Required (Not provided with the kit):
Equipment : Thermocycler, Transilluminator

Reagents : Distilled water, Ethidium Bromide
Other requirements : Crushed ice/Genei cooler,
gloves, micropipette, tips
5
14
Visualisation of DNA fragments: Note:
Since DNA is not naturally coloured, it will not be visible  Read the entire procedure carefully prior to
on the gel. Hence, the gel after electrophoresis, is stained starting the experiment.
with a dye specific to the DNA. Discrete bands are observed  Thaw Template DNA 1 and 2, Primer Mix, 10X Assay
when there is enough DNA material present to bind the dye to Buffer and 10 mM dNTP mix on ice.
make it visible, otherwise the band is not detected. The gel is  For preparation of Agarose Gel, Electrophoresis,
observed against a light background wherein DNA appears as Staining and Destaining refer Agarose Gel
dark coloured bands. Electrophoresis (Page No.10).
Alternatively, an intercalating dye like Ethidium bromide is
added to agarose gel and location of bands determined by Procedure:
examining the gel under UV light. DNA fluoresces. Setting up PCR:
Set up 2 PCR vials with each vial containing one of the two
Note: template DNA samples provided. Add the following reagents
 Ethidium bromide must be handled carefully as it to the PCR tube in the following order.
is a mutagen and a carcinogen.  Template 1.0 µl
 Gels with Ethidum bromide are to be collected in  Primer Mix 4.0 µl
separate plastic bags and shipped to the external  10X Assay Buffer 5.0 µl
waste management agency for incineration as part  10mM dNTP mix 2.0 µl
of hazardous solid waste.  Water 37.5 µl
 Taq DNA Polymerase 0.5 µl (3U/µl)
50.0 µl
2. Mix the contents gently and layer the reaction mix with
20 µl of of mineral oil to prevent evaporation.
Note: Mineral oil need not be added if the
Thermocycler is equipped with a heated lid.
PCR Amplification:
3. Carry out the amplification in a thermocycler for 27
cycles using the following reaction conditions:
Initial Final
Denaturation Denaturation Annealing Extension Extension
94ºC 94°C 60°C 72°C 72°C
2 minutes 30 sec. 30 sec. 45 sec. 7 min.
For 27 cycels
6
13
Analysis on Agarose Gel Electrophoresis of DNA fragments:
4. Following PCR amplification, add 5 µl of Gel Loading Electrophoresis is a technique used to separate charged
Buffer to each of the PCR tubes. molecules. DNA is negatively charged at neutral pH and when
5. Tap the mixture thoroughly and wait for a few electric field is applied across the gel, DNA migrates towards
seconds for the 2 layers to separate. the anode. Migration of DNA through the gel is dependent
upon:
6. Carefully pipette out 5 µl of reaction mixture from
1. Molecular size of DNA
both the tubes (avoiding mineral oil layer) and load
on a to 2% agarose gel. (See page 14). 2. Agarose concentration
3. Conformation of DNA and
7. Load 10 µl of the Ready To Use StepUp™ 100 bp
DNA Ladder provided. Note down the order in which 4. Applied current
the samples have been loaded.
Matrix of agarose gel acts as a molecular sieve through which
8. Electrophorese the samples at 100 volts for 1-2 DNA fragments move on application of electric current. Higher
hours till the tracking dye (bromophenol blue) concentration of agarose gives firmer gels, i.e., spaces
th
reaches 3/4 of the length of the gel. between cross-linked molecules is less and hence smaller
9. Visualize the gel under UV transilluminator. DNA fragments easily crawl through these spaces. As the
length of the DNA increases, it becomes harder for the DNA
to pass through the spaces. Lower concentration of agarose
helps in movements of larger DNA fragments as the spaces
between the cross-linked molecules is more. The progress of
gel electrophoresis is monitored by observing the migration of
a visible dye (tracking dye) through the gel. Two commonly
used dyes are Xylene cyanol and bromophenol blue that
migrate at the same speed as double stranded DNA of size
5000 bp and 300 bp respectively. These tracking dyes are
negatively charged, low molecular weight compounds that are
loaded along with each sample at the start of electrophoresis,
when the tracking dye reaches towards the anode,
electrophoresis is terminated.
7
12
Introduction: Observation:
Agarose gel electrophoresis is a procedure used to Observe the gel under a UV Transilluminator and note down
separate DNA fragments based on their molecular weight and the sizes of the fragment(s) amplified from DNA templates 1
is an intrinsic part of almost all routine experiments carried out and 2 by comparing then with StepUp™ 100 bp DNA ladder
in molecular biology. used as a marker.
The technique consists of 3 basic steps: 1 2 3

 Preparation of agarose gel
 Electrophoresis of the DNA and
 Visualization of DNA fragments
Principle:
Preparation of Agarose Gel: 800 bp
Agarose is a linear polymer extracted from seaweeds. Its
basic structure is shown in figure. 550 bp
HO
CH2O
H Unutilized
primer seen
OH as primer
HO dimer band
Figure: Agarose structure unit Fig 3: Analysis of amplified products from Normal and SNP
DNA on a 2% Agarose Gel.
Purified agarose is a powder insoluble in water or buffer at
room temperature but dissolves on boiling. Molten solution of
agarose is then poured into a mould and allowed to solidify. Lane 1: Normal DNA
As it cools, agarose undergoes polymerization i.e., sugar Lane 2: SNP DNA
polymers cross-link with each other and cause the solution to Lane 3: StepUp™ 100 bp DNA Ladder
gel, the density or pore size of which is determined by
concentration of agarose. Interpretation:
Amplification using SNP type DNA gives 2 bands of 800bp
and 550bp whereas amplification using normal DNA gives
only one band of 800 bp. Hence it can be concluded that DNA
Template 1 is normal and DNA Template 2 is of SNP type.
11 8
Agarose Gel Electrophoresis
9 10

Kt99 Single Nucleotide Polymorphism SNPTK

Uploaded by

Document Information

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Kt99 Single Nucleotide Polymorphism SNPTK

Uploaded by

Copyright:

Available Formats

Single Nucleotide Polymorphism (SNP) GeNei™ Single Nucleotide Polymorphism (SNP) GeNei™

GeNei™ Single Nucleotide

Cat No.: 6109900011730

 Observation & Interpretation 8

Agarose Gel Electrophoresis

An example for SNP is AAGGTTA is altered to ATGGTTA

PCR based SNP detection facilitates scientific research in

In this technique selected regions of a DNA sequence from

No. amplification seen

Fig 1: Normal DNA showing 800 bp fragment amplification

550 bp amplification seen Product Size Cat #

GeNei™ Single Nucleotide 1 EA 6109900011730

Primer 1: Common Forward Primer

Note: Rooom Temperature (RT) not exceed 20°C to 25°C.

Materials Required (Not provided with the kit):

Equipment : Thermocycler, Transilluminator

The technique consists of 3 basic steps: 1 2 3

Agarose Gel Electrophoresis

You might also like