TT A

AB
B LL E
E O
O FF C
ON
N TT E
EN
N TT S
S
CO

Table of Contents

CHAPTER 1

The Basics of PCR and RT-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

The Basics of PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
The Basics of RT-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

CHAPTER 2

Increasing RT-PCR Sensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Isolating High-Quality RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Using RNase H Reverse Transcriptases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Higher RT Incubation Temperatures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Additives to Enhance RT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
RNase H Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Improving Detection of Small Amounts of RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
One-Step vs. Two-Step RT-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3
3
4
5
5
5
6
6

CHAPTER 3

Improving RT-PCR Specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Priming cDNA Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Higher RT Incubation Temperatures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Minimizing Contaminating Genomic DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

7
7
7
7

CHAPTER 4

RT-PCR Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
5 and 3 RACE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Quantifying mRNA Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

CHAPTER 5

Improving PCR Specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Primer Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Primer Annealing Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Touchdown PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Primer Concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Primer Purity and Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Hot Start . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Magnesium Concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Additives to Enhance PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Nested PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

10
10
10
11
11
11
12
13
13
13

CHAPTER 6

Increasing PCR Sensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Template Quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Template Concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Enzyme Choice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

14
14
14
14

CHAPTER 7

Improving Fidelity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Enzymes with Proofreading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Enzyme Mixes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Other Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

15
15
15
15

CHAPTER 8

Other Things to Consider . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Amplifying Long Targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Prevention of Carry-Over Contamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Purification of PCR Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

16
16
16
16

CHAPTER 9

PCR Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Multiplex PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Genotyping with Dinucleotide Repeat Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Tools for Detecting Polymorphisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

17
17
17
17

CHAPTER 10

Troubleshooting Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

CHAPTER 11

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

CHAPTER 12

Related Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

APPENDIX A

Selected Primer Sequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

C
HA
AP
P TT E
ER
R 1
1
CH

The Basics of PCR and RT-PCR

The Basics of PCR

he Polymerase Chain Reaction (PCR)
process uses multiple cycles of template denaturation, primer annealing,
and primer elongation to amplify DNA
sequences (1). It is an exponential process
since amplified products from the previous
cycle serve as templates for the next cycle of

Table 1
Component

Final Concentration

Template

104 10 6 copies of DNA template

Primer 1

0.10.5 M

Primer 2

0.10.5 M

10X Reaction buffer

Magnesium
dNTP mix
Thermostable DNA polymerase

1X
1.03.0 mM
200 M each dNTP
14 units/100 l reaction

TABLE 1. Reaction components.

amplification, making it a highly sensitive

technique for the detection of nucleic acids.
Typically, enough amplified product is
generated after 20 to 30 cycles of PCR so
that it can be visualized on an ethidium
bromide-stained gel. The reaction is composed
of several components (table 1). The template
can include purified genomic or plasmid
DNA; RNA converted to cDNA; or unpurified, crude biological samples such as bacterial colonies or phage plaques. The primers
determine the sequence and the length of the
amplified product. The most frequently
used thermostable polymerase is Taq DNA
polymerase. This enzyme is appropriate for
routine amplifications, but the use of other
thermostable polymerases can enhance results.
Amplification reactions also contain buffer,
deoxynucleotide triphosphates, and magnesium. The magnesium ion concentration
affects enzyme activity, primer annealing,
melting temperature of the template and the
PCR product, fidelity, and primer-dimer
formation (2). How the interaction of these
components, cycling parameters, as well as
other factors contribute to successful PCR
will be discussed in the following chapters.

The Basics of RT-PCR

RT-PCR combines cDNA synthesis from
RNA templates with PCR (figure 1) to provide
a rapid, sensitive method for analyzing gene
expression. RT-PCR is used to detect or
quantify the expression of messages, often
from small amounts of RNA (3,4). In addition,
the technique is used to analyze differential
gene expression or clone cDNAs without
constructing a cDNA library (5,6). RT-PCR is
more sensitive and easier to perform than
other RNA analysis techniques, including
Northern blots, RNase protection assays,
in situ hybridization, and S1 nuclease assays
(7,8).
The template for RT-PCR can be total
RNA or poly(A)+ selected RNA. RT reactions can be primed with random primers,
oligo(dT), or a gene-specific primer (GSP)
using a reverse transcriptase. RT-PCR can
be carried out either in two-step or one-step

formats. In two-step RT-PCR, each step is

performed under optimal conditions. cDNA
synthesis is performed first in RT buffer
and one tenth of the reaction is removed
for PCR. In one-step RT-PCR, reverse transcription and PCR take place sequentially
in a single tube under conditions optimized
for both RT and PCR.
This Guide describes the keys to successful RT-PCR and PCR. High sensitivity
(getting enough product from small samples)
and high specificity (selective amplification
of only the desired product) are the hallmarks
of successful PCR. Optimal RT-PCR and
PCR can be obtained through careful experimental design, including selecting the appropriate enzymes, designing optimal primers,
using different buffers and additives, establishing cycling parameters, and preparing
high quality templates.

Cells or Tissue

RNA Isolation
AAAAAAA(A)n
AAAAAAA(A)n
AAAAAAA(A)n
cDNA Synthesis

gene specific priming

oligo(dT) priming
AAAAAAA(A)n
TTTTTTTT

AAAAAAA(A)n

AAAAAAA(A)n
TTTTTTTT

AAAAAAA(A)n

PCR Amplification

FIGURE 1. RT-PCR overview.

T H E

B A S I C S

O F

P C R

A N D

N6

N6

AAAAAAA(A)n
TTTTTTTT

AAAAAAA(A)n

GSP

random hexamer priming

R T - P C R

N6

N6

N6
N6

AAAAAAA(A)n
N6

N6

N6

N6

AAAAAAA(A)n

AAAAAAA(A)n

C
HA
AP
P TT E
ER
R 2
2
CH

Increasing RT-PCR Sensitivity

TRIZOL Reagent can isolate high-quality RNA from

as little as 100 cells or 1 mg of tissue.

Outline of Procedure for TRIzOL Reagent

RNase inhibitors are often added to RT

reactions to help improve cDNA length and
yield. Add RNase inhibitors to the firststrand reaction in the presence of buffer
and a reducing agent, such as DTT, since
procedures prior to cDNA synthesis that
denature the inhibitor will release bound
RNases that can degrade RNA. Protein RNase
inhibitors protect RNA against degradation
by RNases A, B, and C, but do not protect
against RNases found on the skin. Thus, even
when using these inhibitors be careful not to
introduce RNases from your fingertips.

Total RNA
1

Poly(A)+ RNA
3
4

100 bp
DNA Ladder

susceptible to degradation by trace amounts

of RNases than shorter transcripts (16). To
increase the stability of stored RNA samples,
dissolve the RNA in deionized formamide
and store at 70C. The formamide used for
storage of RNA must not contain impurities
that cause RNA degradation (16). RNA from
pancreas was stable for at least 1 year in formamide. When ready to use the RNA, simply
precipitate it by adding NaCl to 0.2 M
followed by 4 volumes of ethanol. Incubate
3 to 5 min at room temperature and centrifuge
at 10,000 g for 5 min.

100 bp
DNA Ladder

Isolating High-Quality RNA

uccessful cDNA synthesis starts with
high-quality RNA. High-quality RNA
is substantially full length, and does
not contain inhibitors of reverse transcriptases such as EDTA or SDS (9). The quality
of the RNA dictates the maximum amount of
sequence information that can be converted
into cDNA. One popular RNA isolation
protocol is the single-step method which
uses guanidine isothiocyanate/acidic phenol
(10). The TRIZOL Reagent method (see figure
below) is an improvement on this single-step
method and can be used to isolate high quality, undegraded RNA from various cells and
tissues (11,12). The TRIZOL Reagent method
can be used to isolate RNA from as little as
100 cells or 1 mg of tissue (13).
Oligo(dT) selection for poly(A)+ RNA
is typically not necessary. Amplified product
is detectable when either total RNA or
poly(A)+ RNA is used as the starting template (figure 2) (14). In addition, isolating
poly(A)+ RNA may lead to variability of
mRNA enrichment between samples, which
can bias the detection or quantification
of message. However, poly(A)+ RNA may
increase the sensitivity of detection when
analyzing rare messages.
RNA isolated from RNase-rich samples,
such as the pancreas, may need to be stored
in formamide to maintain high quality RNA,
due to the carry-over of trace amounts of
RNase (15). This is especially true for long
term storage. RNA isolated from rat pancreas
and stored in water showed substantial degradation after just 1week, whereas RNA isolated
from rat liver, which contains lower amounts
of RNases, was stable in water for 3 years
(15). In addition, transcripts >4 kb are more

Homogenize sample
in TRIZOL Reagent
377 bp

Separate Phases
(add chloroform)

Panel A

643 bp

Precipitate RNA
Panel B

Wash and Solubilize

Elapsed Time <1 h

I N C R E A S I N G

R T - P C R

S E N S I T I V I T Y

FIGURE 2. Comparison of total RNA and poly(A)+ RNA in RT-PCR.

cDNA was synthesized in duplicate from 5 or 1 g total HeLa RNA
(lanes 1 and 2, respectively) or from 500 ng or 50 ng of poly(A)+
HeLa RNA (lanes 3 and 4, respectively) with oligo(dT) primers and
SUPERSCRIPT II RT. The amplified targets were a 377-bp fragment
near the 5 end of the DNA polymerase mRNA and a 643-bp fragment of the replication protein A mRNA, which are both moderately
abundant targets. One tenth of the cDNA reaction was amplified for
30 cycles using Taq DNA polymerase.

C
HA
AP
P TT E
ER
R 2
2
CH

Using RNase H Reverse Transcriptases

Reverse transcriptases catalyze the conversion
of RNA to cDNA. Both moloney murine
leukemia virus (M-MLV) and avian myleoblastosis virus (AMV) reverse transcriptases
have endogenous RNase H activity in addition to their polymerase activity. The RNase H
activity competes with the polymerase activity
for the hybrid formed between the RNA template and the DNA primer or growing cDNA
strand and degrades the RNA strand of the
RNA:DNA complex (see figure). RNA template that is cleaved by RNase H activity is
no longer an effective substrate for cDNA
synthesis, decreasing both the amount and
size of the cDNA. Therefore, it is advantageous to eliminate or greatly reduce the
RNase H activity of reverse transcriptases.
Figure 3
900

cDNA Synthesized (ng)

800
700

SUPERSCRIPT II RT, an M-MLV RNase

H RT, and THERMOSCRIPT RT, an avian
RNase H reverse transcriptase, yield greater
amounts of cDNA and more full-length
cDNA than M-MLV RT and AMV RT,
respectively (figure 3) (1719). RT-PCR sensitivity can be limited by the amount of cDNA
synthesized. THERMOSCRIPT RT has significantly greater sensitivity than AMV RT
(figure 4) (18). The size of RT-PCR products
is limited by the ability of a reverse transcriptase to synthesize full-length cDNA,
especially when cloning large cDNAs.
SUPERSCRIPT II RT significantly increased
the yield of long RT-PCR products compared to M-MLV RT (figure 5) (20). RNase
H reverse transcriptases also have increased
thermostability, so reactions can be performed
above the normal incubation temperature of 37C
to 42C.

600

THERMOSCRIPT RT
10
1,000

10

AMV RT
100

1,000 RNA (ng)

3.5 kb

FIGURE 4. Effect of RT on sensitivity of RT-PCR. cDNA was synthesized from total HeLa RNA at 50C with oligo(dT) primers using
THERMOSCRIPT RT or AMV RT. PCR was 35 cycles with PLATINUM Taq
DNA Polymerase and primers for human DNA polymerase .
S

M
kb
6.8
5.3

FIGURE 5. Effect of RT on sensitivity in long RT-PCR. The synthesis

of full-length cDNA for human tuberous sclerosis II mRNA (5.3 kb)
and human DNA polymerase mRNA (6.8 kb) was catalyzed by
SUPERSCRIPT II RT (lane S) or M-MLV RT (lane M). cDNA was synthesized from 5 g total HeLa RNA with oligo(dT) primers. Samples
were treated with RNase H and one tenth of the cDNA reaction was
amplified with ELONGASE Enzyme Mix for 35 cycles.

Obstacles Posed by RNase H

500
TTTTTTTT
AAAAAAAA

400

TTTTTTTT
AAAAAAAA

300
TTTTTTTT

200
100
0
AMV RT

THERMOSCRIPT RT

M-MLV RT

SUPERSCRIPT II RT

FIGURE 3. Effect of RT on yield of first-strand cDNA. cDNA was synthesized from 2.5 g (for
THERMOSCRIPT RT and AMV) or 1 g (for SUPERSCRIPT II RT and M-MLV) of a 7.5-kb mRNA using
oligo(dT) primer and 10 Ci of [-32P]dCTP with the recommended synthesis conditions. Total
yield of first-strand ( ) was calculated from TCA precipitation. Full-length cDNA ( ) was
assayed by cutting and counting size-fractionated bands from alkaline agarose gels.

10

1 Kb PLUS
DNA Ladder

Increasing RT-PCR Sensitivity (continued)

I N C R E A S I N G

TTTTTTTT
AAAAAAAA

AA
AA

Effect of RNase H on first-strand cDNA. RNase H degrades RNA in an RNA:DNA complex during cDNA synthesis.
Red arrows represent potential cleavage sites.

R T - P C R

S E N S I T I V I T Y

C
HA
AP
P TT E
ER
R 2
2
CH

Higher RT Incubation Temperatures

Higher RT incubation temperatures (table 2)
can help disrupt RNA secondary structure
and increase the yield of product (18). For
many RNA templates, incubating the RNA
and primer at 65C in the absence of buffers
or salts and quickly chilling on ice eliminates
most secondary structure and allows primer
binding. However, secondary structure may
still exist in some templates even after heat
denaturation. The amplification of products
from these difficult templates can be
improved by using THERMOSCRIPT RT and
performing the RT reactions at higher temperature (figure 6) (18). Higher temperatures
can also improve specificity, especially when
using a gene-specific primer (GSP) for cDNA
synthesis (see page 7). If using a GSP, make
sure the Tm of the primer is as high as the

Table 2
Reverse Transcriptase

Incubation Temperature

AMV

37C45C

M-MLV

37C

SUPERSCRIPT II RT

37C50C

THERMOSCRIPT RT

42C65C*

*RNA begins to hydrolyze at temperatures above 65C. For targets

1 kb, 70C first-stand synthesis temperatures can be used. For
targets >1 kb, use first-strand synthesis temperatures 65C.
TABLE 2. RT incubation temperatures.

elevated RT temperatures, increase specificity

by shifting the RNA/primer mix directly
from the 65C denaturation temperature to
the RT incubation temperature and add prewarmed 2X reaction mix (cDNA synthesis
hot start). This helps prevent intramolecular
base pairing that can occur at lower temperatures. Using a thermal cycler can simplify
the multiple temperature
THERMOSCRIPT RT
AMV RT
1 Kb PLUS
shifts required for RT-PCR.
DNA Ladder
50C
55C
60C
50C
55C
60C
Tth thermostable polymerase, which functions as a
DNA polymerase in the presence of Mg++ and a reverse
transcriptase in the presence
1.5 kb
of Mn++, can also be incubated up to 65C. However,
the presence of Mn++
during PCR reduces fidelity.
This makes Tth polymerase
less suitable for applications
that require highly accurate
amplification, such as cloning
cDNAs. In addition, Tth
FIGURE 6. Effect of temperature on a difficult template. cDNA was synthesized at the indicated temperatures from 10 ng of soybean total RNA with THERMOSCRIPT RT or AMV RT and a
polymerase is a less efficient
gene-specific primer for 18S rRNA. 40 cycles of PCR was performed with one tenth of the
reverse transcriptase, which
cDNA reaction and PLATINUM Taq DNA Polymerase High Fidelity.
reduces sensitivity and, since
planned incubation temperature. Do not use a single enzyme is used for both reverse
oligo(dT) and random primers above 60C; transcription and PCR, a control reaction
random primers require an initial 10 min without RT cannot be used to distinguish
incubation at 25C before increasing the amplified cDNA from amplified contamitemperature to 60C. In addition to using nating genomic DNA.

I N C R E A S I N G

R T - P C R

S E N S I T I V I T Y

Additives to Enhance RT
Additives including glycerol and DMSO can
be added to first-strand synthesis reactions
to help destabilize nucleic acid duplexes and
melt RNA secondary structure. Up to 20%
glycerol or up to 10% DMSO can be used
without effecting SUPERSCRIPT II RT or
M-MLV RT activity (9). AMV RT will also
tolerate up to 20% glycerol without loss of
activity. For maximum RT-PCR sensitivity
in SUPERSCRIPT II RT reactions, add 10%
glycerol and incubate at 45C. If one tenth of
the RT reaction is added to the PCR, the final
concentration of glycerol in the amplification
reaction is 0.4%, which will not inhibit PCR.
RNase H Treatment
Treating cDNA reactions with RNase H prior
to PCR can improve sensitivity. For some
targets, it is thought that the RNA in the
cDNA reaction may prevent binding of the
amplification primers. In these cases, RNase
H treatment can improve sensitivity. RNase
H treatment is often necessary when amplifying longer, full-length cDNA targets, such
S

RNase H
M
A

+ RNase H
M
A

5.3 kb

FIGURE 7. Effect of RNase H treatment on RT-PCR. The synthesis

of full-length cDNA for human tuberous sclerosis II mRNA (5.3 kb)
from 5 g of total HeLa RNA was catalyzed by SUPERSCRIPT II RT (S),
M-MLV RT (M), or AMV RT (A). One half of the reaction was treated
with RNase H for 30 min. An amount of the treated or untreated RT
reaction equivalent to 0.5% of the starting RNA was amplified for 35
cycles with ELONGASE Enzyme Mix.

as the low copy target tuberous sclerosis II

(figure 7) (20). For this difficult target, RNase H
treatment improved the signal from cDNA
generated with either SUPERSCRIPT II RT or
M-MLV RT. For many RT-PCR applications,
RNase H treatment is optional since incubation at 95C for the PCR denaturation step
often hydrolyzes the RNA in the RNA:cDNA
complex.

C
HA
AP
P TT E
ER
R 2
2
CH

Increasing RT-PCR Sensitivity (continued)

A
4

tration of RNase-free glycogen is 250 g/ml

for <50 mg of tissue or <10 6 cultured cells.
The addition of acetylated BSA (figure 8) to
RT reactions performed with SUPERSCRIPT II
RT can increase sensitivity (21). Also, for small
amounts of RNA, reducing the amount of
SUPERSCRIPT II RT and adding 40 units
of RNASEOUT Ribonuclease Inhibitor can
enhance the level of detection. If glycogen is
1

B
4

500 bp

FIGURE 8. Effect of BSA on RT-PCR sensitivity. 50 pg, 5 pg, 500 fg, 50 fg, 5 fg, and 0.5 fg (lanes 1 to 6, respectively) of CAT mRNA transcript
were reverse transcribed without (Panel A) or with (Panel B) 0.5 g of acetylated BSA using SUPERSCRIPT II RT. The no-RT control contained
50 pg of CAT transcript (lane 7). 30 cycles of PCR was performed with Taq DNA polymerase.

used during the RNA isolation, the addition of

BSA or RNase inhibitor to SUPERSCRIPT II RT
reactions is still recommended.
One-Step vs. Two-Step RT-PCR
Two-step RT-PCR is popular and useful for
detecting multiple messages from a single
RNA sample. However, a one-step RT-PCR
method offers other benefits (table 3) (22).
One-step RT-PCR is easier to use when processing large numbers of samples, and helps
minimize carry-over contamination since
tubes do not need to be opened between
cDNA synthesis and amplification. Since the
entire cDNA sample is amplified, one-step
RT-PCR can provide greater sensitivity,
down to 0.1 pg total RNA (figure 9) (22). For
successful one-step RT-PCR, use an antisense
gene-specific primer (GSP) to prime cDNA
synthesis.
1

100 bp
DNA Ladder

Improving Detection
of Small Amounts of RNA
RT-PCR is particularly challenging when only
small amounts of RNA are available. The
addition of glycogen as a carrier during RNA
isolation helps improve the yield from small
samples (13). RNase-free glycogen is added at
the same time TRIZOL Reagent is added.
Glycogen is water soluble and remains in the
aqueous layer with the RNA to aid subsequent
precipitation. The recommended concen-

Table 3
TWO-STEP PROCEDURE

ONE-STEP PROCEDURE

Prime first-strand cDNA with:

Oligo(dT)
Random hexamers
GSP primers

Prime first-strand cDNA with:

GSP primers

Provides:
Flexibility
Choice of primers.
Choice of amplification enzyme.
Ability to optimize for difficult RT-PCR.
Combine with PLATINUM enzymes for higher specificity.
Combine with PLATINUM Pfx DNA Polymerase
for greater fidelity.
Ideal for detecting or quantifying several
messages from a single sample.

Provides:
Convenience
Amplification enzymes premixed with
reverse transcriptase.
Fewer pipetting steps and reduced chances
of contamination.
High sensitivity.
Ideal for analysis of large numbers of samples.
Ideal for quantitative PCR.

Notes on amplification enzymes and product size:

Use Taq DNA Polymerase or PLATINUM Taq DNA Polymerase for products <4 kb.
Use PLATINUM Pfx DNA Polymerase or PLATINUM Taq DNA Polymerase High Fidelity for products <12 kb.
Use ELONGASE Enzyme Mix for products >12 kb.
For one-step RT-PCR, use Taq DNA Polymerase or PLATINUM Taq DNA Polymerase for products <3.5 kb; and use PLATINUM Taq
DNA Polymerase High Fidelity for products <9 kb.
TABLE 3. Comparison of two-step and one-step RT-PCR.

I N C R E A S I N G

R T - P C R

S E N S I T I V I T Y

353 bp

FIGURE 9. Sensitivity of one-step RT-PCR. A -actin fragment

was amplified from 0, 0.1, 1, 10, 102, and 103 pg of total HeLa RNA
(lanes 1 to 6, respectively) using the SUPERSCRIPT One-Step RT-PCR
System. Reactions were incubated at 50C for 30 min; 94C for
2 min; then 40 cycles of 94C for 15 s and 55C for 30 s and 68C
for 90 s; followed by 68C for 5 min. Reactions contained 200 nM of
sense and antisense primer.

C
HA
AP
P TT E
ER
R 3
3
CH

Priming cDNA Synthesis

irst-strand cDNA synthesis reactions
may be primed using three different
methods. The relative specificity of
each method influences the amount and the
variety of the cDNA synthesized.
Random primers are the least specific of
the three methods. The primers anneal to
multiple sites along the entire transcript
to generate short, partial-length cDNAs.
This method is often used to capture 5 end
sequences and make cDNA from RNA
templates with regions of secondary structure
or pause sites that reverse transcriptases cannot copy (2,3). To maximize the size of the
cDNA, the ratio of primers to RNA may need
to be determined empirically for each RNA
preparation. The starting concentration range
is 50 to 250 ng of random primers per 20 l
reaction. Since the majority of the cDNA
synthesized from total RNA with random
primers is ribosomal, poly(A)+ selected RNA
is often used as the template.
Oligo(dT) priming is more specific than
random primers. It hybridizes to the poly(A)
tails found at the 3 ends of most eukaryotic
mRNAs (23). Since poly(A)+ RNA constitutes
approximately 1% to 2% of a total RNA population, the amount and complexity of cDNA
is considerably less than when random
primers are used. Because of its higher specificity, oligo(dT) priming generally does not
require optimization of the primer-to-RNA
ratio or poly(A)+ selection. The use of 0.5 g
of oligo(dT) primer per 20 l reaction is
recommended. Oligo(dT)12-18 is suitable for
many RT-PCR applications. Oligo(dT)20 is
provided with the THERMOSCRIPT RT-PCR
System, since it has greater thermostability
for use at higher RT incubation temperatures.
A gene-specific primer (GSP) is the most
specific primer for the RT step. GSPs are
antisense oligonucleotides that hybridize to

1 Kb PLUS
DNA LADDER

Improving RT-PCR Specificity

specific RNA target sequences, instead of

annealing to entire RNA populations as with
random primers or oligo(dT). The same
rules for designing PCR primers are applied
to the design of the GSP for the RT reaction
(see chapter 5). A GSP can be the same
sequence as the amplification primer which
anneals closest to the 3 end of the message;
or a GSP can be designed to anneal downstream of the reverse amplification primer.
Some targets require the design of more
than one antisense GSP for successful RTPCR, since secondary structure of the RNA
target may prevent primer binding. The use
of 1 pmole antisense GSP in a 20-l firststrand reaction is recommended.
Higher RT Incubation Temperatures
To take full advantage of the specificity
provided by GSPs, use an RT with greater
thermostability. Thermostable RTs can be
incubated at higher temperatures to increase
reaction stringency (17,18). For example, if a
GSP has an annealing temperature of 55C,
the specificity conferred by the GSP is not
fully utilized if the RT reaction is performed
with AMV RT or M-MLV RT under low
stringency at 37C. However, SUPERSCRIPT
II RT and THERMOSCRIPT RT allow for
reaction temperatures of 50C and greater
(table 2, page 5), which can eliminate the
nonspecific products generated at lower
temperatures (figure 10). When maximizing
specificity, add the RNA/primer mix directly
from the 65C denaturation temperature to
the RT incubation temperature and add prewarmed 2X reaction mix (cDNA synthesis
hot start). This helps prevent intramolecular
base pairing that can occur at lower temperatures. Using a thermal cycler can simplify
the multiple temperature shifts required for
RT-PCR.

I M P R O V I N G

R T - P C R

S P E C I F I C I T Y

50C

55C

60C

2 kb

FIGURE 10. Effect of RT reaction temperature on RT-PCR

specificity. cDNA was synthesized from 1 g of HeLa RNA
with THERMOSCRIPT RT and a
GSP designed to anneal to
the human DNA polymerase
mRNA. THERMOSCRIPT RT was
added to a prewarmed reaction
mixture and 35 cycles of PCR
was performed using one tenth
of the cDNA with PLATINUM Taq
DNA Polymerase.

Minimizing Contaminating Genomic DNA

One potential difficulty encountered with
RT-PCR is genomic DNA contamination of
RNA. Using a good RNA isolation technique, such as TRIZOL Reagent, minimizes
the amount of contaminating genomic DNA
in the RNA preparation. To avoid generating
products from genomic DNA, remove
the contaminating DNA by treating RNA
with DNase I, Amplification Grade before
reverse transcription. To terminate the
DNase I digestion, incubate the sample at
65C for 10 min in the presence of 2.0 mM
EDTA. The EDTA chelates the magnesium
and prevents magnesium dependent hydrolysis of the RNA that can occur at higher
temperatures.
To distinguish amplified cDNA from
amplified contaminating genomic DNA,
design primers that anneal in separate
exons. The PCR products derived from
cDNA will be shorter than products amplified from contaminating genomic DNA. In
addition, perform a control experiment without RT for each RNA template to determine
whether a given fragment is of genomic
DNA or cDNA origin. Products generated in
the absence of RT are of genomic origin.

C
HA
AP
P TT E
ER
R 4
4
CH

5 and 3 RACE
apid amplification of cDNA Ends
(RACE) is a procedure to capture
unknown sequences at either the 5
or 3 end of a transcript. Unlike conventional
RT-PCR, which employs two sequencespecific primers, RACE uses one sequencespecific primer and either the poly(A) tail of
mRNAs (3 RACE) or a homopolymeric tail
added to cDNA ends (5 RACE) (figure 11).
RACE has been used for the amplification
and cloning of rare mRNAs (23-25). RACE
products can be cloned, directly sequenced,
used to prepare probes, or combined to generate full-length cDNA (25-27). One method for
joining 5 and 3 RACE products is to use
the sequence information generated by 5
and 3 RACE to design new primers which
will amplify the entire cDNA sequence (26).
The use of RNase H RT and high-fidelity
thermostable polymerases allows higher
fidelity amplifications of longer sequences
to generate full-length cDNA clones.
5 RACE is more challenging and less
specific than RT-PCR applications, since only

Figure 11
mRNA
5

(A)n

GSP1

(A)n

5
3

one of the amplification primers is genespecific. 5 RACE products may be a single

product, multiple products, or even a smear
with no distinguishable products (figure 12)
(28). The quality of the result depends on the
specificity of the GSPs used in first-strand
synthesis and amplification, the specificity of
the anchor primer during amplification, the
complexity and abundance of the target, and
the length of the product. Amplification with
nested primers (up to three rounds of nested
amplification) and using size-selected amplified product as the target in successive rounds
of amplification increases the specificity of 5
RACE (see nested PCR discussion on page 13).
Increasing the RT incubation temperature and
PCR annealing temperature, as well as decreasing the magnesium concentration in the
amplification reaction, can enhance specificity.
5 RACE sensitivity is affected by the RT
used and secondary structure at the 3-end of
the cDNA that may inhibit cDNA tailing.
Incomplete cDNA synthesis lowers the yield
of full-length product and contributes to the
smearing pattern observed
with some targets since shorter
cDNAs are tailed and ampliAnneal first strand
fied. Since SUPERSCRIPT II RT
primer, GSP1,
to mRNA
generates more full-length
cDNA, it can increase the
Copy mRNA into
cDNA with
level of detection of 5 end
SUPERSCRIPT II RT
sequence, especially for tranDegrade RNA
scripts >1 kb (28). Doublewith RNase Mix
Purify cDNA with
stranded
3 termini and hairpin
GLASSMAX Spin
Cartridge
structures impair tailing of
Tail purified cDNA
with dCTP and TdT
cDNA by decreasing the
availability of the 3-OH for
PCR amplify dC-tailed
tailing. The initial incubation
cDNA using the
Abridged Anchor Primer
of the cDNA at 94C followed
and nested GSP2.
by chilling on ice helps to
Reamplify primary
disrupt secondary structure.
PCR product using
AUAP, or UAP, and
Some difficult targets may
nested GSP
require the addition of DMSO
for effective tailing (figure 12)
(28). The tailing enzyme, terTM

3CC...CC

Abridged Anchor Primer

5
GI ... IG
3CC...CC

5
GSP2

UAP
AUAP
5
3

GI ... IG
CC...CC

3
5
nested
GSP

FIGURE 11. Summary of the 5 RACE procedure.

R T - P C R

A P P L I C A T I O N S

1 Kb
DNA Ladder

RT-PCR Applications

kb

2.8
2.7

FIGURE 12. 5 RACE. cDNA was

synthesized from 5 g HeLa total
RNA with SUPERSCRIPT II RT at
45C. 10 l of purified cDNA was
tailed in the presence of 10%
DMSO. 1 l of the tailing reaction
was directly amplified for 40 cycles
with primers for human tuberous
sclerosis and ELONGASE Enzyme
Mix (primary PCR 2.8 kb; lane 1). A
10 l gel plug was removed around
the 2.8-kb band. The DNA was
eluted in 50 l of TE. Nested PCR (30
cycles) was performed using 1 l of
size-selected PCR product (nested
PCR 2.7 kb; lane 2). The gel is
stained with SYBR Green I.

minal deoxynucleotidyl transferase is tolerant

of up to 20% DMSO.
If no products are visible or if only a
smear is visible after nested amplification, a
Southern blot can be used to detect products.
This requires internal sequence information
to use as a probe.
Quantifying mRNA Expression
Quantifying mRNA expression is challenging
RT-PCR, but offers advantages over traditional RNA analysis methods. RT-PCR is
more sensitive than Northern blot or ribonuclease protection analysis and requires less
RNA and sequence information. However,
RT-PCR involves two enzymatic steps that
can contribute to variability in the amount of
RT-PCR product. The amount of RNA converted to cDNA affects yield, but the major
difficulty in quantitative PCR is the exponential nature of PCR, in which small variations between samples translates to large
differences in product yield (29). Two popular

C
HA
AP
P TT E
ER
R 4
4
CH

Relative Fluorescence

For absolute quantification of messages, a

Real-time RT-PCR is non-competitive
and detects the product as it is being formed. standard curve is generated using an in vitro
Several probes can be used to assay the accu- transcribed RNA. The CT value is determined
mulation of product including fluorogenic for different concentrations of the in vitro
5 nuclease probes and Molecular Beacons transcribed RNA. Then the CT values are
(33,34). The first method is based on the 5
plotted against the RNA concentrations to
nuclease activity of Taq DNA polymerase, generate a standard curve for quantifying the
which is used to cleave a hybridization probe level of expression in unknown samples
at the branch point during extension (33). The (figure 13, panel B).
Real-time RT-PCR offers advantages
assay uses probes with two fluorescent labels,
one dye serves as a reporter and the other over competitive RT-PCR. Fluorescence
quenches its signal until the probe is cleaved. monitoring is highly sensitive and highly
Molecular Beacons contain a 5 fluoro- accurate with a linear-dose response over a
phore and 3 quencher. These probes are wide range of target concentrations (figure 13)
designed with a hairpin structure bringing (30). Samples do not require post-amplifithe fluorophore and quencher in close prox- cation analysis and little prior knowledge of
imity to quench fluorescence (34). The stem target abundance is required. Use a two-step
structure is composed of 5 to 7 nucleotides RT-PCR strategy for quantifying multiple
and is GC rich. The outer loop structure is messages in a single RNA sample and use a
composed of 15 to 30 nucleotides and is one-step strategy for added convenience
complementary to the target sequence. When when processing large numbers of samples.
the target sequence is present, the
Panel A
2,000
molecular beacon hybridizes to
1,800
Total HeLa RNA
1,600
the target, relaxing the hairpin
500 ng
50 pg
1,400
50 ng
5 pg
structure and allowing fluores1,200
5 ng
0
500 pg
1,000
cence.
800
For both probes, fluorescent
600
400
measurements are made in real
Fluorescence Threshold
200
time during each cycle of PCR
0
-200
using a thermal cycler coupled to
-400
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44
an optical excitation and detection
Cycle Number
device. The amount of fluoresPanel B
cence released is proportional to
50
45
the amount of PCR product. The
40
35
cycle at which the amount of
30
released fluorescence crosses an
25
20
established baseline fluorescence
15
Slope:
-3.581
10
is known as the threshold cycle
Y-Intercept: 41.56
5
Correlation
Coefficient: 0.993
or CT. The CT value is inversely
0
proportional to the amount of
10
10
10
10
10
10
Starting Quantity (pg total HeLa RNA)
starting target. Thus, high-copyFIGURE 13. Real-time PCR quantitation of retinoblastoma mRNA. RT-PCR of
number samples will have low CT
duplicate samples of total HeLa RNA was performed with the PLATINUM Quantitative
values and low-copy-number
RT-PCR THERMOSCRIPT One-Step System (cDNA synthesis was performed at 60C)
and detected using a FAM-labeled Molecular Beacon probe (400 nM) on an ABI
samples will have high CT values
Prism 7700. Panel A. Relative fluorescence intensity of duplicate samples during
(figure 13).
PCR. Panel B. Standard curve.
Threshold Cycle (CT)

methods for quantifying mRNA abundance

are competitive, quantitative PCR and realtime PCR (4,30).
In competitive PCR, an exogenous RNA
transcript (internal standard RNA) is added
before the RT step to control for sample-tosample variation. The internal standard RNA
is reverse transcribed and amplified with
the target to control for differences in the
efficiency of cDNA conversion and amplification. Quantitation is performed by coamplification of the specific target sequence
together with known concentrations of the
internal standard RNA. The abundance of the
target is determined by comparing the signal
obtained for the internal standard to the signal
for the target.
The internal standard RNA shares the
same primer recognition sites with the target, but is a different size. Several methods
exist to generate internal standards (31). One
of the simplest methods involves installing
the primer recognition sites on nonspecific
spacer DNA by PCR (31). This product can
be cloned into a vector carrying the T7 or
SP6 RNA polymerase promoter, or the RNA
promoter sequences can be included on
the forward primer in order to install the
sequences by PCR (32). Then, RNA standards
can be generated by in vitro transcription.
Competitive, quantitative PCR can be performed in one-step RT-PCR using a GSP or
performed in a two-step reaction using a
GSP or oligo(dT). Oligo(dT) sequence can
be installed by adding poly(dT) sequences
to the reverse primer (32).
Competitive RT-PCR is an end-point
analysis that requires the amplification
efficiency of the target and the internal standard to be equal. Some prior knowledge of
the amount of starting target is necessary
to establish a concentration range for the
internal standard. For accurate quantitation,
reactions containing more standard than target and less standard than target are required.

R T - P C R

A P P L I C A T I O N S

C
HA
AP
P TT E
ER
R 5
5
CH

Improving PCR Specificity

Primer Design
areful primer design is one of the
most important aspects of PCR. The
ideal primer pair anneals to unique
sequences that flank the target and not
to other sequences in the sample. Poorly
designed primers may amplify other, nontarget sequences. The following guidelines
describe the desirable characteristics of a
primer sequence that increase specificity:
Typical primers are 18 to 24 nucleotides in
length. The primer needs to be long enough
for the sequence to be unique and to reduce
the probability of the sequence being found
at non-target sites. However, primers greater
than 24 nucleotides do not confer greater
specificity. Longer sequences can hybridize
with some mismatching, which decreases
specificity, and hybridize slower than shorter
sequences, which may decrease yield (2).
Select primers that are 40% to 60% GC or
mirror the GC content of the template.
Design primers with G or C residues in the
5 and central regions. This increases the
primers stability and confers hybridization
stability with the target sequence.
Avoid complementary sequences at the
3 end of primer pairs. This prevents amplification from the primers themselves to
form primer-dimers.
Avoid a GC-rich 3 end. Design primers
to contain 3 As or Ts within the last 5
nucleotides (35).
Avoid mismatches at the 3 end. The last 3
nucleotide needs to anneal to the template
for the polymerase to catalyze extension
(36).
Avoid sequences with the potential to form
internal secondary structure. This destabilizes primer annealing.
Additional sequences that are not present
on the target, such as restriction sites and
promoter sequences, can be added to the 5
end of a primer without affecting specificity.
These sequences are not included when estimating the Tm of a primer. However, check

10

Careful primer design

is one of the most important
aspects of PCR. Poorly designed
primers may amplify other,
non-target sequences.

low enough to guarantee efficient annealing

of the primer to the target, but high enough
to minimize nonspecific binding. Reasonable annealing temperatures range from
55C to 70C. Annealing temperatures are
generally set about 5C below the Tm of the
primers.
There are several formulas for estimating
the Tm (38-40). Table 4 lists two of the commonly used formulas for determining a
primers Tm. The first formula was derived for
hybridization in high salt (1 M) and is valid
for primers <18 bases. The second formula
estimates Tm based on GC content and salt
concentration. The most reliable method for
determining the Tm of a primer is the nearestneighbor analysis (38). This method predicts
the hybridization stability of a primer from

these regions for complementarity and internal

secondary structures.
Sometimes only limited sequence information is available for primer design. For
example, if only the amino acid sequence
is known, degenerate primers are designed.
Degenerate primers are a mix of different
sequences representing all possible bases
coding for a single amino
Table 4
acid. To improve specificity,
Simple Formula (39) (valid for primers <18 bases)
minimize the degeneracy conTm = 4C (G + C) + 2C (A + T)
sulting codon usage tables for
base preference in different
Tm for Oligonucleotides (40) (dependent on salt concentration)
organisms (2). In addition,
Tm = 81.5 + 16.6 (log10[Na+]) + 0.41 (%G + C) 675/n
deoxyinosine may be used in
Where n = number of bases and [Na+] = monovalent (Na+ or K+) cations
places where more than one
TABLE 4. Formulas to estimate Tm .
base is possible. Inosine pairs
with all bases and will lower the annealing the primary sequence and the identity of the
temperature of the primer. Do not include neighboring bases. Most computer programs
degenerate bases at the 3 end of the primer, use nearest-neighbor analysis.
The Tm can vary significantly depending
since annealing of the last three bases on the
3 end can be enough to initiate PCR at the on the formula used and the primer sequence.
wrong sites. Use higher primer concentrations Since most formulas provide an estimated
(1 M to 3 M) because many of the primers Tm value, the annealing temperature is only a
in the degenerate mixture are not specific for starting point. Specificity can be increased
by analyzing several reactions with increasthe target (37).
ingly higher annealing temperatures. Begin
at 5C below the estimated Tm and increase
Primer Annealing Temperature
Another important parameter for primers is the annealing temperature in 2C increthe melting temperature (Tm). This is the ments. Higher annealing temperatures can
temperature at which 50% of the primer and reduce the formation of both primer-dimer
its complementary sequence are present in a and nonspecific products. For best results,
duplex DNA molecule. The Tm is necessary both primers need to have a similar Tm.
to establish an annealing temperature for Primer pairs whose Tm varies by more than
PCR. Ideally, the annealing temperature is 5C exhibit greater mispriming due to the

I M P R O V I N G

P C R

S P E C I F I C I T Y

C
HA
AP
P TT E
ER
R 5
5
CH

use of lower annealing temperatures during

cycling. If two primers have a different Tms,
set the annealing temperature 5C below
the lowest Tm. Or to increase specificity,
perform 5 cycles at the annealing temperature established by the primer with the
higher Tm and then the remaining cycles at
the annealing temperature established by
lower Tm. This allows some copies of the
target to be generated under more stringent
conditions.
Touchdown PCR
Touchdown PCR increases sensitivity by
using stringent annealing conditions during
the early cycles of PCR (41). Cycling begins
with annealing temperature approximately
5C above the estimated Tm. It is incrementally decreased by 1C to 2C every cycle
until the annealing temperature is about
5C below the Tm. Only targets with the
greatest homology will be amplified. These
products will continue to be amplified and
will compete out nonspecific products
amplified in later cycles. Touchdown PCR
is useful in applications where the degree of
homology between the target sequences and
the primer are unknown, such as AFLP
DNA fingerprinting.
Primer Concentration
Primer concentration can affect specificity.
Optimal primer concentration typically falls
between 0.1 to 0.5 M. Higher concentrations of primer may result in the amplification of nonspecific products.
To determine primer concentration, read
the optical density at 260 nm (OD260). Then,
using Beers Law (formula 1) calculate the
concentration by using the absorbance and
the reciprocal of the millimolar extinction
coefficient (nmol/OD). The millimolar
extinction coefficient can be calculated
using formula 2 (42). Unlike large doublestranded DNA molecules where an averaged

Primer Purity and Stability

Standard purity Custom Primers are suffiFormula 1
cient for most PCR applications (table 5).
Concentration= A 260 dilution factor the reciprocal of the
extinction coefficient conversion factors
With Life Technologies PARALLEL ARRAY
SYNTHESIS technology, desalting is not necExample: To calculate the concentration of an oligonucleotide
essary.
The benzoyl and isobutyryl groups
in 1 ml, measure the A260 of 10 l of the oligonucleotide in
990 l of water (1:100 dilution). If the A260 = 0.14 OD and
removed by desalting are found in lower
the oligonucleotide has a reciprocal extinction coefficient
amounts with PARALLEL ARRAY SYNTHESIS
of 4.9 nmol/OD, the concentration would be calculated as
follows:
technology, compared to other methods, and
Concentration = 0.14 OD 100 4.9 nmol 1 mol 103 ml
thus do not interfere with PCR. Some appliml
OD
103 nmol
L
cations require purification to remove any
= 69 M
less than full-length sequences generated
during synthesis. These truncated sequences
Formula 2
are generated because DNA synthesis chemMillimolar Extinction Coefficient of Oligonucleotide =
istry is not 100% efficient. It is a cyclical
A(15.2) + C(7.05) + G(12.01) + T(8.4) at pH 8.0
process in which DNA is synthesized 35
Where A, C, G, and T are the number of dAs, dCs, dGs,
and dTs (35).
using chemical reactions that are repeated
for each base added. Failures can occur during
extinction coefficient can be used, the use of any cycle. Longer primers, especially primers
the extinction coefficient calculated for >50 bases, have a greater portion of truncated
the primer is essential to accurately deter- sequences and may require purification.
Primer yield is affected by the efficiency
mine the concentration (42). This is because
primers are short and the base composition of the synthesis chemistry and by the method
can vary greatly. Both the extinction coeffi- of purification. Life Technologies guarantees
cient (OD/mol) and the reciprocal of the the total oligonucleotide yield as a minimum
extinction coefficient (mol/OD) are pro- number of OD units (table 6, page 12). Custom
vided on the GIBCO BRL Custom Primer Primers are shipped lyophilized. It is best to
certificate of analysis. In addition, do not reconstitute primers in TE [10 mM Tris-HCl
estimate primer concentration on ethidium (pH 8.0), 1 mM EDTA] to bring the final
bromide-stained gels using oligonucleotides concentration to 100 M. TE is better than
as standards, since the staining capability of deionized water since the pH of the water is
the standard and the primer can vary greatly often slightly acidic and can cause hydrolysis
depending on their sequence (43).
of the oligonucleotide.
The stability of the primer
Table 5
depends on storage conditions.
Application
Minimum Suggested Purity
Store lyophilized and reconstiPCR
Standard
tuted primers at 20C. Primers
First-strand cDNA synthesis for RT-PCR
Standard
reconstituted in TE at 10 M
AFLP technology
Standard
are stable for >6 months at
PCR using primers with 5 sequences
Cartridge
20C, but only <1 week when
(restriction endonuclease sites, RNA polymerase promoter sites, etc)
stored at room temperature
PCR primers >50 bases
PAGE
(15C to 30C). Lyophilized
Cycle sequencing
Standard
Isothermal sequencing
Desalted
primers are stable >1 year at
Site-directed mutagenesis
Cartridge
20C, and up to 2 months
CFLP technology
Desalted
when stored at room temperature (15C to 30C).
TABLE 5. Minimum recommended primer purity for PCR applications.

I M P R O V I N G

P C R

S P E C I F I C I T Y

11

C
HA
AP
P TT E
ER
R 5
5
CH

ation temperature. Most manual hot-start

methods involve the delayed addition of Taq
DNA polymerase, which can be cumbersome especially for high-throughput applications (46). Other hot-start methods use wax
barriers to encapsulate an essential component, such as magnesium or enzyme, or to
physically separate reaction components,
such as template and buffer, from each other.
Melting of the wax during thermal cycling
releases and mixes all of the components
together. Like manual hot-start methods,
wax barrier methods can be cumbersome,
prone to contamination and unreliable in
high-throughput applications.
PLATINUM DNA Polymerases are convenient and highly-effective for automatic
hot-start PCR (figure 14). PLATINUM Taq
DNA Polymerase is comprised of recombinant Taq DNA polymerase complexed with
monoclonal antibodies to Taq DNA polymerase (47). The antibodies prevent enzyme
activity during PCR set-up and even during
prolonged incubations at room temperature.

Table 6
Minimum Yield (OD) for Different Primer Purities*
Number of Bases

Synthesis Scale

Standard/
Desalted

<20

50 nmole
200 nmole
1 mole
10 mole
50 nmole
200 nmole
1 mole
10 mole

2
8
20
200
5
20
50
500

20

Cartridge

HPLC

PAGE

2
8
20
NA
2
10
25
NA

NA
3
10
100
NA
3
15
150

NA
1
3
30
NA
1
5
50

Note: For primers >50 bases, PAGE purification is recommended and HPLC is not an option.
NA is not available.
*These yields are seen with the following 5 modifications: biotin, fluorescein (FITC), rhodamine, primary amines (NH2), phosphate (PO4),
HEX, TET, FAM, and phosphorothioates (S-Oligos). Other modifications may have slightly lower yields.
TABLE 6. Minimum oligonucleotide yield for the various purification methods.

inactivate enzyme activity and therefore does

not completely eliminate the amplification of
nonspecific products.
Hot start delays DNA synthesis by withholding one of the essential components
until the thermal cycler reaches the denatur-

12

Taq DNA polymerase is released into the

reaction after incubation at 94C during the
denaturation step, restoring full polymerase
activity. In comparison to chemically modified Taq DNA polymerase for hot-start PCR,
PLATINUM enzymes do not require pro-

I M P R O V I N G

P C R

S P E C I F I C I T Y

1 Kb
DNA Ladder

Hot Start
Hot-start PCR is one of the most important
methods, in addition to good primer design,
for increasing PCR specificity. Even though
the optimal extension temperature for Taq
DNA polymerase is approximately 72C, the
polymerase has activity at room temperature
(44). Thus, nonspecific products are often
generated during PCR set-up and at the start
of thermal cycling when reactions are briefly
incubated at temperatures well below the
annealing temperature (45,46). Once these
nonspecific products are formed they can be
efficiently amplified. Hot-start PCR is particularly effective when the sites available for
designing primers are limited due to the
location of genetic elements, such as sitedirected mutagenesis, expression cloning, or
the construction and manipulation of genetic
elements for DNA engineering.
A popular method that limits Taq DNA
polymerase activity is to set-up amplification
reactions on ice and place them into a preheated thermal cycler. This method is simple
and inexpensive, but does not completely

100 bp
DNA Ladder

Improving PCR Specificity (continued)

4.1 kb

114 bp

Panel A

Panel B

FIGURE 14. Improved specificity with PLATINUM Taq DNA Polymerase. Panel A. Detection of cloned HIV DNA in human genomic
DNA. 1,000 copies of plasmid DNA with the HIV gag region was
mixed with 100 ng of genomic DNA and amplified with primers to the
gag region. Lane 1. Taq DNA polymerase with room temperature
assembly. Lane 2. Taq DNA polymerase with manual hot start by
addition of enzyme at 94C. Lane 3. PLATINUM Taq DNA Polymerase
with room temperature assembly. Panel B. Amplification of 4.1 kb of
human -globin from 100 ng of human genomic DNA. Lane 1. Taq
DNA polymerase with room temperature assembly. Lane 2.Taq DNA
polymerase with assembly on ice and placed in a preheated (80C)
thermal cycler. Lane 3. PLATINUM Taq DNA Polymerase with room temperature assembly.

longed incubation at 94C (10 to 15 min) to

activate the polymerase. Greater than 90%
of Taq DNA polymerase activity is restored
in 2 min at 94C with PLATINUM Taq DNA
Polymerase (48).
Magnesium Concentration
Magnesium affects several aspects of PCR
including DNA polymerase activity, which
can affect yield; and primer annealing, which
can affect specificity. The dNTPs and
template bind magnesium and reduce the
amount of free magnesium available for
enzyme activity. The optimal magnesium
concentration varies for each primer pair and
template, but the starting concentration in
typical PCR containing 200 M dNTPs is
1.5 mM (Note: for real-time quantitative
PCR use 3 to 5 mM magnesium with fluorescent probes) (2). Higher concentrations of
free magnesium can result in greater yield,
but can also increase nonspecific amplification and reduce fidelity (49,50). To determine the best concentration, perform a
magnesium titration using from 1 mM to
3 mM in 0.5 mM increments. To reduce the

C
HA
AP
P TT E
ER
R 5
5
CH

Additives to Enhance PCR

Optimization of the annealing temperature,
primer design, and magnesium concentrations is adequate to achieve high specificity
amplification of many targets. However,
some targets, including those with a highGC content, require additional measures.
Additives that affect DNA melting temperature provide another method for improving
product specificity and yield (figure 16).
Complete denaturation of the template is

magnesium optimization and works with

PLATINUM Taq DNA Polymerase (figure 16)
and PLATINUM Pfx DNA Polymerase. Thus,
combining PLATINUM technology and additives enhances specificity while minimizing
the need for the third approach-magnesium
optimization. For best results, optimize the
concentration of the additives (figure 17),
especially DMSO, formamide, and glycerol
that inhibit Taq DNA polymerase (46,52).

Nested PCR
Sequential rounds of amplification using
nested primers can improve specificity and
sensitivity (3). The first round is a standard
amplification of 15 to 20 cycles. A small
aliquot of the initial amplification is diluted
1:100 to 1:1,000 and added to the second
round of amplifica(mM MgCl2)
(mM MgCl2)
tion with 15 to 20
1.0
1.4
1.8
2.2
2.6
3.0
1.0
1.4
1.8
2.2
2.6
3.0
cycles. Alternatively,
the initial amplification product can
2.8 kb
be size selected by
gel purification. Two
different primer sets
are used for the two
rounds of amplification. The second
Panel B
Panel A
amplification uses a
FIGURE 15. Broader magnesium range with PLATINUM Taq DNA Polymerase. A 2.8-kb region of the
nested set of primers
human -globin gene was amplified from 100 ng of human genomic DNA. Panel A.Taq DNA polymerase
with assembly on ice and placed in a preheated (80C) thermal cycler. Panel B. PLATINUM Taq DNA Polythat bind to the target
merase with room temperature assembly.
just inside the first
required to obtain the best results. Addi- set of primers. The chance of amplifying
tionally, secondary structure can prevent multiple targets is reduced with nested
primer binding and enzymatic elongation. PCR since fewer targets will be complePCR additives, including formamide, mentary to both sets of primers; whereas
DMSO, glycerol, betaine, and PCRX performing the same total number of cycles
Enhancer Solution can enhance amplifi- (30 to 40) with the same set of primers
cation (51-53). Their proposed mechanism is often amplifies nonspecific targets. Nested
to lower the melting temperature, thereby PCR can increase the sensitivity from limited
aiding primer annealing and helping the amounts of target, such as amplifying a rare
DNA polymerase extend through regions message, and increase the specificity of
of secondary structure (53). PCRX Solution more challenging PCR applications such
has additional benefits. It requires less as 5 RACE.

I M P R O V I N G

P C R

S P E C I F I C I T Y

156 bp
62% GC

Panel A

Panel B

FIGURE 16. Improved specificity with PCRx Enhancer Solution.

A 156-bp fragment (62% GC content), was amplified from 100 ng of
human genomic DNA using PLATINUM Taq DNA Polymerase. MgCl2
was titrated (1.0, 1.5, 2.0, 2.5 mM, lanes 1 to 4 respectively) using:
standard PCR buffer (panel A) or PCRx Amplification Buffer with 1X
PCRx Enhancer Solution (panel B). Cycling was 35 cycles of 94C for
30 s; 60C for 30 s, and 68C for 1 min.
100 bp
DNA Ladder

need for magnesium optimization, use

PLATINUM Taq DNA Polymerase. PLATINUM
Taq DNA Polymerase functions over a
broader range of magnesium concentration
than Taq DNA polymerase so less optimization is required (figure 15) (47).

0X

1X 2X 2.5X 3X 3.5X 4X

149 bp
78% GC

FIGURE 17. Titration of PCRx Enhancer Solution. A 149-bp (78%

GC) trinucleotide repeat-containing sequence was amplified from
100 ng of human genomic DNA with PLATINUM Taq DNA Polymerase
in 1X PCRx Amplification Buffer with varying amounts of PCRx
Enhancer Solution (0 to 4X).

13

C
HA
AP
P TT E
ER
R 6
6
CH

Template Quality
emplate quality affects product yield. A
number of contaminants found in DNA
preparations can inhibit PCR (46).
Reagents such as SDS, which are used in standard genomic DNA preparations, can inhibit
amplification reactions at concentrations as
low as 0.01%. Newer methods for isolation of
high-quality genomic DNA include DNAZOL
Reagents (53), which are guanidine-detergent
lysing solutions, and the FTA Products,
which is a matrix-bound method for
the storage and purification of DNA from
blood and other biological samples (table 7) (55).

1 Kb PLUS
DNA LADDER

Increasing PCR Sensitivity

Enzyme Choice
In addition to using high-quality template
DNA, the choice of polymerase also affects
yield (see table on page 22). PLATINUM polymerases provide better yield than other polymerases because they prevent amplification
of nonspecific product during PCR set-up
(figure 18). For high-sensitivity PCR of long
products (up to 12 kb), choose an enzyme
mix, preferably in a PLATINUM format, such
as PLATINUM Taq DNA Polymerase High
Fidelity. This enzyme combines the benefits of
PLATINUM technology with those of enzyme
mixes (Taq DNA Polymerase mixed with a
proofreading polymerase).

14

5
kb

8.4

4.1

Taq DNA Polymerase

Template Concentration
The amount of starting template is important
for obtaining good product yields. For most
amplifications, 10 4 to 10 6 starting target
molecules allows sufficient amplification
to visualize the product on an ethidium bromide-stained gel (2). The optimal amount of
template required depends on the size of the
genome (table 8) (56). For example, 100 ng to
1 g of human genomic DNA, correlating to
3 10 4 to 3 105 molecules, is sufficient to
detect a PCR product from a single-copy gene.
For plasmid DNA, which is much smaller,
the amount of DNA added to PCR is in the
picogram range.

PLATINUM Taq DNA Polymerase

PLATINUM Taq DNA Polymerase High Fidelity

FIGURE 18. Amplification with different thermostable polymerases. Human blood was spotted on
FTA Cards. DNA was amplified directly from 1 to 3 mm punches of washed FTA Cards in 50 l using Taq
DNA polymerase (panel A), PLATINUM Taq DNA Polymerase (panel B), and PLATINUM Taq DNA Polymerase
High Fidelity (panel C). Amplified products were 4.1, 5.2, 7.5, 8.0, and 8.4 kb in lanes 1 to 5, respectively.

Table 7
Method

Description

Proteinase K/phenol extraction

Classic method

DNAZOL Reagents

Fast, phenol-free DNA isolation

FTA Products

Matrix-bound purification and storage

TABLE 7. Methods for genomic DNA isolation.

Table 8
Target Molecules/g
of Genomic DNA

Amount of DNA (g)

for ~10 5 Molecules

Genomic DNA

Size (bp)*

E. coli

4.7 10 6

1.8 108

0.001

Saccharomyces cerevisiae

2.0 107

4.5 107

0.01

Arabidopsis thaliana

7.0 107

1.3 107

0.01

Drosophila melanogaster

1.6 108

6.6 105

0.5

Homo sapiens

2.8 109

3.2 105

1.0

Xenopus laevis

2.9 109

3.1 105

1.0

Mus musculus

3.3 109

2.7 105

1.0

Zea mays

1.5 1010

6.0 104

2.0

2.69 103

3.4 1011

1 10 6

pUC 18 plasmid DNA

* Haploid genome size

TABLE 8. Correlation of genome size and number of molecules.

I N C R E A S I N G

P C R

S E N S I T I V I T Y

C
HA
AP
P TT E
ER
R 7
7
CH

1 Kb PLUS
DNA LADDER

1 Kb PLUS
DNA LADDER

Enzymes with Proofreading

CR applications involving cloning
and sequence analysis, expression
of PCR products, or site-directed
mutagenesis require high-fidelity PCR.
Taq DNA polymerase is considered a lowfidelity polymerase since it lacks 3 to 5
exonuclease (proofreading) activity. The use
of thermostable polymerases with 3 exonuclease activity improve fidelity (57,58).
However, these polymerases can give lower
yields than Taq DNA polymerase. PLATINUM
Pfx DNA Polymerase has significantly better
fidelity than Taq DNA polymerase and
offers the advantage of high yield and specificity of PLATINUM products (figure 19) (59).

1 Kb PLUS
DNA LADDER

Improving Fidelity

1.1 kb

Panel A

Panel B

Panel C

FIGURE 19. High sensitivity and specificity with PLATINUM Pfx DNA Polymerase. Genomic DNA (100, 50, 10, 5, 1, and 0 ng, lanes 1 to 6)
was amplified with primers for a 1.1-kb fragment of human thrombospondin for 35 cycles. Panel A. 1 unit of PLATINUM Pfx DNA Polymerase
with room temperature set-up. Panel B. 2.5 units of PfuTurbo DNA Polymerase with set-up on ice. Panel C. 1 unit of Taq DNA Polymerase
with set-up on ice.

Enzyme Mixes
Mixing Taq DNA polymerase with a second
polymerase with 3 exonuclease activity
provides greater fidelity than Taq DNA
polymerase alone and allows for higher
yield and amplification of longer templates.
The GIBCO BRL high fidelity enzyme mix,
PLATINUM Taq DNA Polymerase High
Fidelity has 6-times greater fidelity than Taq
DNA polymerase alone and can amplify up
to 12 kb.
Other Parameters
Besides enzyme, high dNTP or magnesium
concentrations can reduce fidelity. Decreasing
the concentration of dNTPs from 200 M
to 25-50 M can increase accuracy. If the
concentration is not the same for all four
nucleotides, the fidelity will be effected.
Performing fewer cycles of PCR also can
help increase fidelity, since the probability of
a mutation increases with increasing cycle
number and product length.

I M P R O V I N G

F I D E L I T Y

15

C
HA
AP
P TT E
ER
R 8
8
CH

Other Things to Consider

The Concert Rapid PCR Purification

System purifies PCR fragments
from 80 bp to 10 kb.
amplification of targets up to 30 kb (61).
PLATINUM Pfx DNA Polymerase (has 35
exonuclease activity) can also amplify longer
products (12 kb).
In addition to choosing the correct thermostable polymerase, the amplification of
longer products requires changes to the
extension times, denaturation times, and
buffer pH of the standard protocol. Increase
extension times to 1 min/kb to allow the
polymerase to complete synthesis. Typically
the extension temperature is lowered to
68C for more effective long PCR (60,61).
Since the elongation times are long, up to
20 min for a 20-kb target, a buffer with a
higher initial pH is used. If the pH falls
below pH 7.0, the DNA can depurinate.
To protect the template against damage,
minimize the denaturation time at 94C to
30 s or less during each cycle and limit the
preamplification denaturation time to 94C
for 1 min. Primers are designed in the same
manner as those used in a standard protocol,
with primers 18 to 24 bases giving good
product yields (60,61).

16

Prevention of Carry-Over Contamination

PCR is susceptible to contamination problems because it is a sensitive amplification
technique. Small amounts of contaminating
DNA from an exogenous source can be
amplified along with the desired template.
A common source of contamination occurs
when previously amplified products are
introduced into new amplification reactions.
This is called carry-over contamination.
Purified DNA from other samples and cloned
DNA can also be sources of contamination.
Carry-over contamination can be minimized by using good laboratory procedures
during PCR. Use aerosol-resistant tips to
prevent aerosols from reaching the pipette
barrel. Designate separate areas for PCR
sample set-up and post-amplification analysis
and change gloves before preparing new
reactions. Always perform a negative control,
without template, to check for contamination. Use premixed reaction components
instead of adding each reagent to individual
reactions.
One method to prevent carry-over contamination uses uracil DNA glycosylase
(UDG) (62). This enzyme (also known as
uracil-N-glycosylase or UNG) removes
uracil residues from DNA. Substituting
deoxyuracil for thymine in amplification
reactions allows previously amplified products to be distinguished from template DNA.
Since the previously amplified products
are susceptible to UDG, newly assembled
reactions are treated with UDG before PCR
to destroy carry-over products.
Purification of PCR Products
Residual reaction components including
primers, nucleotides, and thermostable polymerases can inhibit cloning, sequencing, and
labeling reactions. Thus, it is often necessary
to purify PCR products before further manipulation. Clean-up processes involving multiple
phenol:chloroform extractions followed by
PEG precipitation (63) or isopropanol pre-

O T H E R

T H I N G S

T O

C O N S I D E R

cipitation (2) are effective but time consuming and can result in product losses. The
CONCERT Rapid PCR Purification System
purifies PCR products from reaction components in 10 min. It is effective for a wide
range of PCR fragments, from 80 bp to
10 kb, and provides efficient recovery of up
to 95% of the original sample (figure 20) (64).
Some PCR products may require purification, from nonspecific PCR products that
can interfere with cloning or sequencing,
by gel electrophoresis. The desired product
is purified from the agarose using the
CONCERT Gel Extraction System, a silicabased technology for rapid isolation of DNA
fragments from gels.
When cloning PCR products by restriction
endonucleases, TAQUENCH PCR Cloning
Enhancer provides an alternative to the
clean-up of PCR products. If 3-recessed
termini are generated by digestion, residual
Taq DNA polymerase and dNTPs from the
PCR can fill in the 3-recessed termini (65).
This lowers the cloning efficiency and generates clones ligated in the improper reading
frame. However, the addition of TAQUENCH
Enhancer before restriction digestion minimizes Taq DNA polymerase activity for efficient cloning (65).
High DNA
MASS Ladder

Amplifying Long Targets

hen amplifying targets >5 kb, use
an enzyme or enzyme mix for
long PCR to obtain the best yield
(see table on page 22 and figure 18 on page 14).
Taq DNA polymerase does not efficiently
amplify longer targets (>5 kb), presumably
because it lacks 35exonuclease activity
and cannot correct dNTP misincorporations
(60). The elongation rate from a mismatch is
greatly reduced, which decreases the yield of
longer products. Mixing Taq DNA polymerase
with a thermostable DNA polymerase containing 35 exonuclease activity can allow

1
A

2
B

B
kb

0.7

primers

FIGURE 20. Purification of PCR fragments following amplification. Completed PCRs containing ~1 g of amplified product were
spiked with 1.2 g of primer (lane 1) or 3.5 g of primer (lane 2).
Primers were 36 to 40 bases. Spiked reactions were purified, and
aliquots of the eluate before (lane A) and after (lane B) purification
were analyzed by agarose electrophoresis.

C
HA
AP
P TT E
ER
R 9
CH
9

PCR Applications

Multiplex PCR
n multiplex PCR, multiple primer sets
are used simultaneously to amplify
several different loci. This complex
PCR often results in low product yield and
requires higher concentrations of magnesium
(67). A false negative result may be obtained

Tools for Detecting Polymorphisms

Amplified Restriction Fragment Polymorphism (AFLP) technology is a technique for
fingerprinting genomic DNA from plants
and microorganisms (figure 23) (68,69). RFLP,
AP-PCR, and RAPD are also common
DNA fingerprinting techniques. RFLP is a
hybridization-based method
primer (nM) 250
250
200
200
100
200
200
100
template (ng) 100
100
250
500
500
250
500
500
which is time consuming and
M
M
bp
requires a large amount of
genomic DNA. AP-PCR and
RAPD are faster, PCR-based
methods, but both are sensitive to reaction conditions
547
459
416
which can affect the repro360
ducibility of the techniques.
268
AFLP technology combines the reliability of RFLP
and the convenience of a
PCR-based fingerprinting
Panel B
Panel A
method for a more robust
FIGURE 21. PLATINUM Taq DNA Polymerase provides greater sensitivity for multiplex
and informative method.
PCR. Multiplex amplification of the dystrophin gene was performed with 5 different primer
DNA fingerprints generated
sets from human genomic DNA withTaq DNA polymerase (panel A) or PLATINUM Taq DNA
Polymerase (panel B). Using the indicated primer and template concentration, products
with AFLP technology can
ranging from 268 bp to 547 bp were amplified from human genomic DNA.
be used as a tool to identify a
if reactions are not optimized correctly. The specific DNA sample or to assess the simiincreased robustness of PLATINUM Taq DNA larity between samples (figure 24) (69).
A number of parameters influence AFLP
Polymerase over a broad range of magnesium concentrations increases success in results, including the number of selective
nucleotides in the primers (69). The number
multiplex reactions (figure 21).
of amplified bands decreases when the
Genotyping with
number of selective nucleotides increases.
Dinucleotide Repeat Markers
Larger genomes require more selective
Determining the size of the amplified dinucle- nucleotides to amplify an appropriate
otide repeat region can be challenging since number of DNAs, since more restriction
Taq DNA polymerase often adds a nontem- fragments are generated from digesting a
plated nucleotide to PCR products. The frac- larger genome.
tion of products in an amplification reaction
The template quality can also affect
containing an extra nucleotide is sequence AFLP results. Poor quality template may
and primer dependent and can vary greatly. inhibit restriction endonucleases, resulting
However, PLATINUM GENOTYPE Tsp DNA in incomplete digestion and fewer amplified
Polymerase exhibits minimal nontemplated bands visible on the gel (69). However, the
nucleotide addition to help increase the accu- AFLP technique is tolerant of variation in
racy of allele size determination (figure 22) template concentration, with no difference
(66). Simply substitute PLATINUM GENOTYPE observed with amounts of DNA between
Tsp DNA Polymerase for Taq DNA poly- 100 ng to 5 g.
merase in these amplifications.

Figure 21
Nucleotides

Fluorescence Intensity

150

155

2,500
2,000
1,500
1,000
500
0

Taq DNA Polymerase

(66% n + 1)

2,500
2,000
1,500
1,000
500
0

PLATINUM GENOTYPE Tsp

DNA Polymerase
(0% n + 1)

A P P L I C A T I O N S

n
n+1

FIGURE 22. Comparison of DNA polymerases in genotyping

of dinucleotide repeat markers. Amplification of nga106 locus of
Arabidopsis thaliana generated withTaq DNA polymerase (66% n + 1)
or PLATINUM GENOTYPE Tsp DNA Polymerase (0% n + 1).

Figure 23
5
3

GAATTC
CTTAAG

TTAA
AATT

3
5

+Eco R I
Mse I
AATTC
G
TTAA
Eco R I adapter

P C R

160

n+1
n

T
AAT
+Eco R I adapter
Mse I adapter
TA

Mse I adapter
primer +1

A
AATTCN
TTAAGN

NTTA
NAAT
C

GTTA
CAAT
AAC

preselective
amplification with
Eco R I primer +A
Mse I primer +C
primer +3

AAC
AATTCA
TTAAGT

selective amplification
with primers +3
AATTCAAC
TTAAGTTG

TTGTTA
AACAAT

denaturing polyacrylamide gel electrophoresis

Mse I adapter sequences

Eco R I adapter sequences

FIGURE 23. Schematic of the AFLP Analysis System.

1 2 3 4 5 6 7 8 9

FIGURE 24. A typical AFLP

fingerprint. The polymorphism of
soybean ecotypes was performed
using the AFLP System I and the
selective primers Eco R I + ACC
and Mse I + CAA. Ecotypes were
Holladay; Morgan; Hytest; Essex;
Bass; T135; P88; PI88788; P83;
PI83495; P90; PI90763; lanes 1
to 9, respectively.

17

C
HA
AP
P TT E
ER
R 1
10
0
CH

Troubleshooting Guide

Problem
RT-PCR Sensitivity:
Little or no RT-PCR product
visible after agarose gel analysis.

PCR Sensitivity:
Little or no PCR product
visible after agarose gel analysis.

18

Possible Cause

Suggested Solution

RNA was degraded

Analyze RNA on a denaturing gel before use to verify integrity.

Use good aseptic technique for RNA isolation.
Process tissue immediately after removal from animal.
Store RNA in 100% formamide (15).
If using placental RNase inhibitor, do not heat >45C or use >pH 8.0 or inhibitor
may release any bound RNases. Also, DTT must be present when the RNase
inhibitor is added at 0.8 mM DTT.

RNA contained an inhibitor of RT

Remove inhibitor by ethanol precipitation of the RNA. Include a 70% (v/v) ethanol
wash of the RNA pellet. Glycogen (0.25 g to 0.4 g/l) can be included to aid in
RNA recovery for small samples.
Inhibitors of RT include: SDS, EDTA, glycerol, sodium pyrophosphate, spermidine,
formamide, and guanidinium salts (9).
Test for inhibitors by mixing a control RNA with the sample and comparing yields
to control RNA reaction.

Polysaccharide coprecipitation of RNA

Precipitate RNA with lithium chloride to remove polysaccharides.

Primer used for first-strand cDNA

synthesis did not anneal well

Be sure annealing temperature is appropriate for your primer. For random hexamers, a 10 min incubation at 25C is recommended before incubating at reaction
temperature.
For gene-specific primers (GSP), try another GSP or switch to oligo(dT) or random
hexamers.
Make sure GSP is the antisense sequence.

Not enough starting RNA

Increase the amount of RNA.

For <50 ng RNA, use 0.1 g to 0.5 g acetylated BSA in first-strand cDNA
synthesis (21).

RNA template had high secondary structure

Denature/anneal RNA and primers in the absence of salts and buffer.

Raise the RT reaction temperature up to 50C for SUPERSCRIPT II RT or up to 65C
for THERMOSCRIPT RT.
Note: Do not use oligo(dT) as a primer over 60C and choose a GSP that will
anneal at your reaction temperature. For RT-PCR products >1 kb, keep reaction
temperature 65C.
Note: Do not use M-MLV RT above 37C.
Use random hexamers in the first-strand reaction if full-length cDNA is not needed.

The primers or template are sensitive to

remaining RNA template

RNase H treat first-strand cDNA before PCR (14).

Target not expressed in tissue analyzed

Try a different target or tissue.

PCR did not work

For two-step RT-PCR, do not use more than 1/5 of the RT reaction in the PCR step.

Poor PCR primer design

Avoid complementary sequences at the 3 end of primers. Avoid sequences that

can form internal hairpin structures. Design primers with similar Tms.

DNA contains inhibitors

Reagents such as DMSO, SDS, and formamide can inhibit Taq DNA polymerase.
If inhibitor contamination is suspected, ethanol precipitate the DNA sample.

GC-rich template

For templates >50% GC content, use PCRx Enhancer Solution.

Template concentration is too low

Start with 104 copies of the target sequence to obtain a signal in 25 to 30 cycles.

Magnesium concentration is too low

Determine the optimal magnesium concentration for each template and primer
pair by performing a reaction series from 1 mM to 3 mM in 0.5 mM increments.
Note: Use 3 mM to 5 mM magnesium for real-time quantitative PCR.

T R O U B L E S H O O T I N G

G U I D E

C
HA
AP
P TT E
ER
R 1
10
0
CH

Problem
PCR Sensitivity:
Little or no PCR product
visible after agarose gel analysis.
(continued)

RT-PCR Specificity:
Unexpected bands
after gel analysis

PCR Specificity:
Unexpected bands
after agarose gel analysis

Possible Cause

Suggested Solution

Annealing temperature is too high

Use the equations listed in table 4 (page 10) to estimate the Tm and set the annealing
temperature 5C below the Tm. Since these equations estimate Tm values, the true
annealing temperature may actually be higher or lower.

Primer concentration is too low

Optimal primer concentration is between 0.1 M to 0.5 M. To accurately determine primer concentration, read the optical density at 260 nm (OD260). Then,
calculate the concentration using the absorbance and the extinction coefficient
(see page 11).

Nonspecific annealing of primers

to templates

Use a GSP instead of random hexamers or oligo(dT) for first-strand synthesis.

Poor GSP design

Follow the same rules as described for amplification primers.

Genomic DNA contamination of RNA

Treat RNA with DNase I, Amplification Grade (see page 7). Check for DNA contamination with a control reaction without RT.

Primer-dimer formation

Design primers without complementary sequences at the 3 ends.

Nonspecific annealing of primers

to template

Increase the annealing temperature in 2C to 5C increments and minimize the

annealing time.

Try a GSP that allows cDNA synthesis at high temperatures.

Use higher annealing temperatures for the first few cycles, followed by lower
annealing temperatures.
Use PLATINUM Taq DNA Polymerase for automatic hot-start PCR (47).
Avoid 2 or 3 dGs or dCs at the 3 end of primers.

PCR Fidelity:
PCR induced errors found
in the product sequence

Magnesium concentration is too high

Optimize magnesium concentration for each template and primer combination.

Primer mispriming due to amplification

from complex templates.

Use nested primers or touchdown PCR.

Contaminating DNA from an

exogenous source

Use aerosol-resistant tips and UDG (see page 16).

Primer binding sites are inaccessible

due to secondary structure

For templates >50% GC content use (1X-3X) PCRX Enhancer Solution.

Polymerase has low fidelity

Use a proofreading thermostable polymerase such as PLATINUM Pfx DNA Polymerase.

Too many cycles

Reduce cycle number.

The concentration of all four

deoxynucleotides is not equal

Prepare a new deoxynucleotide mix and ensure that the concentration of all four
nucleotides is equal or use a prepared mix.

T R O U B L E S H O O T I N G

G U I D E

19

C
HA
AP
P TT E
ER
R 1
11
1
CH

References

1. Saiki, R.K., Scharf, S., Faloona, F., Mullis, K.B., Horn, G.T.,
Erlich, H.A., and Arnheim, N. (1985) Science 230, 1350.

27. Fitscher, B.A., Riedel, H.D., Young, K.C., and Stremmel, W.

(1998) Biochem. Biophys. Acta 1443, 381.

2. Innis, M.A., Gelfand, D.H., Sinsky, J.J., and White, T.J.

(1990) PCR Protocols, A Guide to Methods and
Applications. Academic Press, San Diego, California.

28. Fox, D.K., Westfall, B., Nathan, M., Hughes, A.J.,

Rashtchian, A., and Schuster, D.M. (1996) FOCUS 18, 33.

3. Dieffenbach, C.W. and Dveksler, G.S. (1995) PCR Primer:

A Laboratory Manual. Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, New York.
4. Siebert, P.D. and Larrick, J.W. (1991) Nature 359, 557.
5. Liang, P. and Pardee, A.B. (1992) Science 257, 967.
6. Frohman, M.A. (1993) Methods Enzymol. 218, 340.
7. Foley, K.P., Leonard, M.W., and Engel, J.D. (1993)
Trends in Genetics 9, 380.
8. Mocharla, H., Mocharla, R., and Hodes, M.E. (1990)
Gene 93, 271.

9. Gerard, G.F. (1995) FOCUS 16, 102.

10. Chomczynski, P. and Sacchi, N. (1987) Anal. Biochem.
162, 156.
11. Chomczynski, P. (1993) BioTechniques 15, 532.
12. Simms, D., Cizdziel, P.E., and Chomczynski, P. (1994)
FOCUS 15, 99.
13. Bracete, A.M. and Fox, D.K. (1999) FOCUS 21, 38.
14. Fox, D.K. and Nathan, M. (1997) FOCUS 19, 48.
15. Bracete, A.M., Fox, D.K., and Simms, D. (1998)
FOCUS 20, 82.

29. Freeman, W.M., Walker, S.J., and Vrana, K.E. (1999)

BioTechniques 26, 112.
30. Heid, C.A., Stevens, J., Livak, K.J., and Williams, P.M.
(1996) Genome Res. 6, 986.
31. Zimmerman, K. and Mannhalter, J.W. (1996)
BioTechniques 21, 268.
32. Vanden Heuvel, J.P., Tyson, F.L., and Bell, D.A. (1993)
BioTechniques 14, 395.
33. Holland, P.M., Abramson, R.D., Watson, R., and Gelfand,
D.H. (1991) Proc. Natl. Acad. Sci. USA 88, 7276.
34. Tyagi, S., Bratu, D.P., and Kramer, F.R. (1998)
Nature Biotechnology 16, 49.
35. Lear, W., McDonel, M., Kashyap, S., and Boer, P. (1995)
BioTechniques 18, 84.
36. Kwok, S., Kellogg, D.E., McKinney, N., Spasic, D.,
Goda, L., Levenson, C., and Sinsky, J. (1990)
Nucleic Acids Res. 18, 999.
37. White, B.A. (1993) PCR Protocols, Current Methods and
Applications. Humana Press, Totowa, New Jersey.

54. Ally, A.H. and Chomczynski, P. (1995) FOCUS 17, 70.

55. Hansen, P. and Blakesley, R. (1998) FOCUS 20, 72.
56. Brown, T.A. (1991) Molecular Biology Labfax. Academic
Press, Inc., San Diego, California.
57. Cline, J., Braman, J.C., and Hogrefe, H.H. (1996)
Nucleic Acids Res. 24, 3546.
58. Tindall, K.R. and Kunkel, T.A. (1988) Biochemistry 27, 6008.
59. Westfall, B., Sitaraman, K., Lee, J.E., Borman, J., and
Rashtchian, A. (1999) FOCUS 21, 46.
60. Westfall, B., Sitaraman, K., Berninger, M., and Mertz, L.
(1993) FOCUS 17, 62.
61. Cheng, S., Fockler, C., Barnes, W.M., and Higuchi, R.
(1994) Proc. Natl. Acad. Sci. USA 91, 5695.
62. Longo, M.C., Berninger, M.S., and Hartley, J.L. (1990)
Gene 93, 125.
63. Paithankar, K. R. and Prasand, K.S. (1991)
Nucleic Acids Res. 19, 1346.
64. Young, A., Xu, L., Goldsborough, M., and Blakesley, R.
(1999) FOCUS 21, 11.
65. Fox, D.K., Nathan, M., Natarajan, P., Bracete, A.M., and
Mertz, L. (1998) FOCUS 20, 15.
66. Jordan, H., Darfler, M., and Solus, J. (1999) FOCUS 21, 4.

38. Breslauer, K.J., Frank, R., Blocker, H., and Marky, L.A.
(1986) Proc. Natl. Acad. Sci. USA 83, 3746.

67. Chamberlain, J.S., Gibbs, R.A., Ranier, J.E., Nguyen, P.N.,

and Caskey, C.T. (1988) Nucleic Acids Res. 16, 11141.

39. Nelson, T. and Brutlag, D. (1979) Methods Enzymol. 68, 41.

68. Lin, J.J., Kuo, J., and Ma, J. (1996) Nucleic Acids
Res. 24, 3649.

16. Nakajima, D., Nakayama, M., and Ohara, O. (1998)

FOCUS 20, 80.

40. Rychlik, W., Spencer, W.J., and Rhoads, R.E. (1990)

Nucleic Acids Res. 18, 6409.

17. Gerard, G.F., DAlessio, J.M., and Kotewicz, M.L. (1989)

FOCUS 11, 66.

41. Don, R.H., Cox, P.T., Wainwright, B.J., Baker, K., and
Mattick, J.S. (1991) Nucleic Acids Res. 19, 4008.

70. Raff, T., van der Giet, M., Endemann, D., Wiederholt, T.,
and Paul, M. (1997) BioTechniques 23, 456.

42. Fox, D.K. (1998) FOCUS 20, 84.

71. Khiri, H., Reyneir, P., Peyrol, N., Lerique, B., Torresani, J.,
and Planells, R. (1996) Mol. Cell Probes 10, 201.

18. Schwabe, W., Lee, J.E., Nathan, M., Xu, R.H., Sitaraman,
K., Smith, M., Potter, R.J., Rosenthal, K., Rashtchian, A.,
and Gerard, G.F. (1998) FOCUS 20, 30.
19. Schuster, D.M., Darfler, M., Lee, J.E., and Rashtchian, A.
(1998) FOCUS 20, 34.

43. Sewall, A., Natarajan, P., and Fox, D.K. (1999) FOCUS 21, 2.
44. Li, H., Cui, X., and Arnheim, N. (1990) Proc. Natl. Acad.
Sci. USA 87, 4580.

69. Lin, J.J. and Kuo, J. (1997) FOCUS 17, 66.

72. Tokunaga, K., Taniguchi, H., Shimizu, M., and Sakiyama, S.

(1986) Nucleic Acids Res. 14, 2829.

45. Chou, Q., Russell, M., Birch, D., Raymond, J., and Bloch,
W. (1992) Nucleic Acids Res. 20, 1717.

73. Strehlau, J., Pavlakis, M., Lipman, M., Shapiro, M.,

Vasconcellos, L., Harmon, W., and Strom, T. (1997)
Proc. Natl. Acad. Sci. USA 94, 695.

22. Lee, H.E., Sitaraman, K., Schuster, D., and Rashtchian, A.

(1997) FOCUS 19, 39.

46. Erlich, H.A. (1989) PCR Technology, Principles and

Applications for DNA Amplification. Stockton Press,
New York, New York.

74. Arcari, P., Martinelli, R., and Salvatore, F. (1984)

Nucleic Acids Res. 12, 9179.

23. Frohman, M.A., Dush, M.K., and Martin, G.R. (1988)

Proc. Nat. Acad. Sci. USA 85, 8998.

47. Westfall, B., Sitaraman, K., Solus, J., Hughes, J., and
Rashtchian, A. (1997) FOCUS 19, 46.

24. Schuster, D.M., Buchman, G.W., and Rashtchian, A. (1992)

FOCUS 14, 46.

48. Westfall, B., Darfler, M., Xu, R., and Rashtchian, A. (1998)
FOCUS 20, 17.

25. Loh, Y., Elliott, J.F., Cwirla, S., Lanier, L.L., and Davis, M.M.
(1989) Science 243, 217.

49. Eckert, K.A. and Kunkel, T.A. (1990) Nucleic Acids

Res. 18, 3739.

26. Cowell, I. and Austin, C.A. (1997) Methods in Molecular

Biology: cDNA Library Protocols. Humana Press, Totawa,
New Jersey.

50. Williams, J.F. (1989) BioTechniques 7, 762.

20. Nathan, M., Mertz, L., and Fox, D.K. (1995) FOCUS 17, 78.
21. Nathan, M. and Fox, D.K. (1997) FOCUS 19, 50.

51. Pomp, D. and Medrano, J.F. (1991) BioTechniques 10, 58.

52. Varadaraj, K. and Skinner, D.M. (1994) Gene 140, 1.
53. Baskaran, N., Kandpal, R., Bhargava, A., Glynn, M.,
Bale, A., and Weissman, S. (1996) Genome Res. 6, 633.

20

R E F E R E N C E S

75. Zhao, J., Araki, N., and Nishimoto, S.K. (1995)

Gene 155, 159.
76. Fort, P.H., Marty, L., Piechaczyk, M., el Sabrouty, S.,
Dani, C.H., Jeanteur, P.H., and Blanchard, J.M. (1985)
Nucleic Acids Res. 13, 1431.

C
HA
AP
P TT E
ER
R 1
11
1
CH

Locating FOCUS References

Many of the References listed on

the previous page are FOCUS articles.
These articles contain many details
related to protocols to obtain data
presented in this Guide.

Locating Previous FOCUS Articles

FOCUS Volumes 16 Current Issue can be found on our web site at www.lifetech.com. Several
of the Classic FOCUS articles (most requested by you through Technical Services) can also be
found on the web. Other articles, can be obtained from Literature Fulfillment through your local
Life Technologies Office.

Viewing a FOCUS Article from our Web Site

Finding the Article:
When you click on the FOCUS journal on the Home Page, the table of contents for the
most recent issue will appear with old issues in the left box. (Older issues not displayed
there are found by clicking Archives). There is also a search box in the upper left which
will allow you to do a full text search of FOCUS articles.
Viewing the Article:
To view the article, you will need the latest Adobe Acrobat Reader. If you do not currently
have the Acrobat Reader, you can download it for free at:
www.adobe.com/products/acrobat/readstep.html
There is a link to this site in the paragraph at the top of the FOCUS table of contents.
Follow the procedure to download the Acrobat Reader. There are 2 stages to the process:
1. Follow Adobes directions for downloading the proper file to your local drive.
(Note file location when saving.)
2. Locate file you saved and install the reader on your computer.
Note: To install the reader, just double click on the file on your local drive and follow
the prompts. The reader is accessible to you for future use. You will not need to
download the reader again, unless you remove the program from your computer.
Now you can locate the FOCUS article, click on it, and it will open on your screen.

Cumulative FOCUS Subject Index

There are 2 ways to find articles on a specific technique:
1. The web site has a built-in subject index for the articles on the web. Just use the Search
box at the top of the FOCUS section of the web.
2. A Cumulative Subject Index is available for volumes 5-21. You can find this document on
our web site or contact Literature Fulfillment through your local Life Technologies Office.
Instructions to Authors
Looking for information on submitting articles to FOCUS ?
Check out the Instructions to Authors file in the FOCUS section of our web site.

L O C A T I N G

F O C U S

R E F E R E N C E S

21

C
HA
AP
P TT E
ER
R 1
12
2
CH

Related Products

Comparison of Enzymes Used in PCR

Product
Taq DNA Polymerase
Taq PCRX DNA Polymerase
PCR SUPERMIX
PLATINUM Taq DNA Polymerase
PLATINUM Taq PCRX DNA Polymerase
PLATINUM PCR SUPERMIX
PLATINUM Quantitative PCR SUPERMIXUDG

Size
<5 kb
<5 kb
<5 kb
<5 kb
<5 kb
<5 kb
<5 kb
<12 kb

PLATINUM Pfx DNA Polymerase

PLATINUM Taq DNA Polymerase High Fidelity

<12 kb
<12 kb

PLATINUM GENOTYPE Tsp DNA Polymerase

<0.5 kb

ELONGASE Enzyme Mix

<30 kb

PCR SUPERMIX High Fidelity

Yield

Specificity

Fidelity

* Contains PCRX Enhancer System, ideal for high-GC content or other problematic templates.

PCR Enzymes
Product

Cat. No.

Size

Taq DNA Polymerase, Native*

18038-018
18038-042
18038-067

100 units
500 units
1,500 units

Taq DNA Polymerase, Recombinant*,1

10342-053
10342-020
10342-046

100 units
500 units
1,500 units
(3 500 units)

PLATINUM Taq DNA Polymerase*,2,14

10966-018
10966-026
10966-034

100 units
250 units
500 units

PLATINUM Pfx DNA Polymerase*,2,14

11708-013
11708-021
11708-039

100 units
250 units
500 units

PLATINUM Taq DNA Polymerase High Fidelity*,2,14

11304-022
11304-029

100 units
500 units

Taq PCRX DNA Polymerase*,2,12

11508-017

500 units

11509-015

500 units

PLATINUM GENOTYPE Tsp DNA Polymerase*,2,12,14,15

11448-024
11448-032

250 units
2,500 units

PLATINUM Taq Antibody *,2,14

10965-010
10965-028

100 units
250 units

,1

(Includes PLATINUM Pfx DNA Polymerase, PCRX Enhancer Solution, 10X Pfx Amplification Buffer, and MgSO4)

(Consists of Taq DNA Polymerase, PCRX Enhancer System, 10X PCR Buffer, and MgCl2)

PLATINUM Taq PCRX DNA Polymerase*,2,12,14

(Consists of PLATINUM Taq DNA Polymerase, PCRX Enhancer System, 10X PCR Buffer, and MgCl2)

22

R E L A T E D

P R O D U C T S

C
HA
AP
P TT E
ER
R 1
12
2
CH

PCR Enzymes (continued)

Product

Cat. No.

Size

PCR SUPERMIX*,2,12

10572-014

100 reactions

PLATINUM PCR SUPERMIX*,2,12,14

11306-016

100 reactions

PCR SUPERMIX High Fidelity*,2,12

10790-020

100 reactions

PLATINUM Quantitative PCR SUPERMIX-UDG*,2, 9,12,14, 24

11730-017
11730-025

100 reactions
500 reactions

PCR Reagent System*,2

10198-018

100 reactions

ELONGASE Amplification System*,1,2

10481-018

100 reactions

ELONGASE Enzyme Mix*,1,2

10480-010

100 reactions

RT-PCR
Product

Cat. No.

SUPERSCRIPT II RNase H Reverse Transcriptase

Size

18064-014
18064-022
18064-071

2,000 units
10,000 units
4 10,000 units

SUPERSCRIPT RNase H Reverse Transcriptase 5

18053-017

10,000 units

SUPERSCRIPT First-Strand Synthesis System for RT-PCR 5

11904-018

50 reactions

10928-018
10928-026

25 reactions
100 reactions

10928-034
10928-042

25 reactions
100 reactions

11922-010
11922-028

25 reactions
100 reactions

11146-024
11146-016

25 reactions
100 reactions

THERMOSCRIPT RT-PCR System and PLATINUM Taq DNA Polymerase*,2,13,14

11146-057
11146-032

25 reactions
100 reactions

THERMOSCRIPT RT-PCR System and PLATINUM Taq DNA Polymerase High Fidelity*,2,13,14

11146-040

100 reactions

PLATINUM Quantitative RT-PCR THERMOSCRIPT One-Step System*,2,13,14,24

11731-015
11731-023

100 reactions
500 reactions

M-MLV Reverse Transcriptase

28025-013
28025-021

40,000 units
200,000 units

RT-PCR Primer and Control Set

10929-016

20 reactions

Ribonuclease H

18021-014
18021-071

30 units
120 units

DNase I, Amplification Grade

18068-015

100 units

RNASEOUT Recombinant Ribonuclease Inhibitor

10777-019

5,000 units

DEPC-treated Water

10813-012

4 1.25 ml

5 RACE System for Rapid Amplification of cDNA Ends, Version 2.0 4,5

18374-058

10 reactions

3 RACE System for Rapid Amplification of cDNA End 4,5

18373-019

20 reactions

(Includes oligo(dT)12-18, random hexamers, SUPERSCRIPT II RT, RNASEOUT RNase Inhibitor 10X RT buffer,
Mg solution, 10 mM dNTP mix, E. coli RNase H, control RNA and primers, and DEPC treated water)

SUPERSCRIPT One-Step RT-PCR System*,2, 5

(Includes SUPERSCRIPT II RT/Taq DNA Polymerase enzyme mix, 2X reaction buffer, Mg solution)

SUPERSCRIPT One-Step RT-PCR System with PLATINUM Taq DNA Polymerase 2, 5,14
(Includes SUPERSCRIPT II RT/PLATINUM Taq DNA Polymerase Mix, 2X Reaction Mix, and MgSO4)

SUPERSCRIPT One-Step RT-PCR for Long Templates 2, 5,14

(Includes SUPERSCRIPT II RT/PLATINUM Taq DNA Polymerase High Fidelity Mix, 2X Reaction Mix, and MgSO4)

THERMOSCRIPT RT-PCR System*,2,13

(Includes THERMOSCRIPT RT, 5X cDNA Synthesis Buffer, oligo(dT)20, random hexamers, 0.1 M DTT,
10 mM dNTP Mix, RNASEOUT RNase Inhibitor, DEPC-treated water, E. coli RNase H)

(Includes THERMOSCRIPT PLUS/PLATINUM Taq Mix, 2X THERMOSCRIPT Reaction Mix, and MgSO4)

(Includes HeLa RNA and -actin primers)

R E L A T E D

P R O D U C T S

23

C
HA
AP
P TT E
ER
R 1
12
2
CH

Related Products (continued)

Reaction Components
Product

Cat. No.

Size

GIBCO BRL Custom Primers

Please Inquire

10 mM dNTP Nucleotide Mix 4

18427-013

100 l

100 mM dNTP Nucleotide Set 4

10297-018

4 250 l

Nucleic Acid Purification

Product

Cat. No.

Size

TRIZOL Reagent

15596-026
15596-018

100 ml
200 ml

TRIZOL LS Reagent

10296-010
10296-028

100 ml
200 ml

DNAZOL Reagent

10503-027
10503-035

100 ml
200 ml

DNAZOL BD Reagent

10974-020
10974-038

100 ml
200 ml

Plant DNAZOL Reagent

10978-021

100 ml

Buffered-Saturated Phenol

15513-039

100 ml

Proteinase K

25530-015

100 mg

FTA Cards 4,16

10786-010

100/pkg

FTA GeneCard 4,16

10786-036

100/pkg

FTA Purification Reagent 4,16

10876-019

250 ml

CONCERT Rapid PCR Purification System

11458-015
11458-023

50 reactions
250 reactions

CONCERT Rapid Gel Extraction System

11456-019
11456-027

50 reactions
250 reactions

TAQUENCH PCR Cloning Enhancer 4,12,14

11265-014
11265-022

100 units
250 units

Other Applications
Product
AFLP Analysis System I (Genome size of 5 10 to 6 10 )

9 17

Cat. No.

Size

10544-013

each

10717-015

each

11352-010

each

10822-013

125 l

(Contains AFLP Core Reagent Kit and AFLP Starter Primer Kit)

AFLP Analysis System II (Genome size of 1 108 to 5 108)17

(Contains AFLP Core Reagent Kit and AFLP Small Genome Primer Kit)

AFLP System for Microorganisms (Designed for bacteria and fungi)17

(Contains AFLP Core Reagent Kit and AFLP Microorganism Primer Kit)

AFLP Non-radioactive Probe

24

R E L A T E D

P R O D U C T S

C
HA
AP
P TT E
ER
R 1
12
2
CH

Related PCR Products

Product

Cat. No.

100 bp DNA Ladder

15628-019

Size
50 g

1 Kb PLUS DNA LADDER

10787-018

250 g

0.2-ml PCR Tubes

10795-011

1,000 tubes/bag

0.5-ml PCR Tubes

10795-037

1,000 tubes/bag

0.2-ml PCR Tubes with Flat Caps

10795-151

1,000 tubes/bag

0.2-ml PCR Tubes with Attached Strip Caps

10795-144

125 strips/bag

Thin-wall Reaction Tubes with Wax Beads

Product

Cat. No.

Size

HOTSTART 50 Storage and Reaction Tubes (20 to 50 l reactions)

10331-015

96 tubes

HOTSTART 100 Storage and Reaction Tubes (50 to 100 l reactions)

10332-013

96 tubes

HOTSTART Micro 20 Storage and Reaction Tubes (15 to 25 l reactions)

10467-017

96 tubes

HOTSTART Micro 50 Storage and Reaction Tubes (25 to 50 l reactions)

10468-015

96 tubes

Silicone Oil

10890-010

4 ml

Uracil DNA Glycosylase 9

18054-015

100 units

ART, Aerosol Resistant Tips, Brand Pipet Tips

Product

Cat. No.

Size

ART REACH

10670-016

960 tips/pkg

ART 20P

14862-015

1,000 tips/pkg

ART 20E

14862-023

960 tips/pkg

ART 200

14867-014

960 tips/pkg

ART 1000

14868-012

800 tips/pkg

U.S. Academic/TECH-LINE: (800) 828-6686

U.S. Government Orders/TECH-LINE: (888) 584-8929
U.S. Industrial Orders/TECH-LINE: (800) 874-4226
Internet: www.lifetech.com
These products are for laboratory research use only and are not intended for human or
animal diagnostic, therapeutic, or other clinical uses, unless otherwise stated.
ABI PRISM is a registered trademark of Perkin Elmer Corporation.
AFLP is a registered trademark of Keygene n.v.
ART, Aerosol Resistant Tips, and REACH are marks of Molecular Bio-Products, Inc.
CFLP is a trademark of Third Wave Technologies, Inc.,
DNAZOL and TRIZOL are registered trademarks of Molecular Research Center, Inc.,
FTA is a registered trademark of Flinders Technologies PTY Ltd.
HOTSTART 50, HOTSTART 100, HOTSTART Micro 20, and HOTSTART Micro 50 are
registered trademarks of Molecular Bio-Products, Inc.
PfuTurbo is a trademark of Stratagene.
SYBR is a registered trademark of Molecular Probes, Inc.
TaqMan is a registered trademark of Roche Molecular Systems, Inc.

R E L A T E D

Purchase of this product is accompanied by a limited license to use it in the Polymerase Chain Reaction (PCR) process for research and development in conjunction
with a thermal cycler whose use in the automated performance of the PCR process
is covered by the up-front license fee, either by payment to Perkin-Elmer or as
purchased, i.e., an authorized thermal cycler. This product is sold under licensing
arrangements with F. Hoffmann-La Roche Ltd, Roche Molecular Systems, Inc., and
The Perkin-Elmer Corporation.

P R O D U C T S

25

C
HA
AP
P TT E
ER
R 1
12
2
CH

Related Products (continued)

Limited Label Licenses

1
A license under U.S. Patents 4,683,202, 4,683,195, 4,965,188, and 5,075,216 or
their foreign counterparts, owned by Hoffmann-La Roche Inc. and F. HoffmannLa Roche Ltd (Roche), has an up-front fee component and a running-loyalty
component. The purchase price of this product includes limited, nontransferable
rights under the running-royalty component to us only this amount of the product
to practice the polymerase chain reaction (PCR) and related processes described
in said patents solely for the research and development activities of the purchaser
when this product is used in conjunction with a thermal cycler whose use is
covered by the up-front fee component. Rights to the up-front fee component
must be obtained by the end user in order to have a complete license. These rights
under the up-front fee component may be purchased from Perkin-Elmer or
obtained by purchasing an Authorized Thermal Cycler. No right to perform of
offer commercial services of any kind using PCR including, without limitation,
reporting the results of the purchasers activities for a fee or other commercial
consideration, is hereby granted by implication or estoppel. Further on
purchasing licenses to practice the PCR Process may be obtained by contacting
the Director of Licensing at the Perkin-Elmer Corporation, 850 Lincoln Centre
Drive, Foster City, California 94404 or at Roche Molecular Systems, Inc., 1145
Atlantic Avenue, Alameda, California 94501.
A license under U.S. Patent 4,683,202, 4,683,195, and 4,965,188 or their foreign
counterparts, owned by Hoffman-La Roche Inc. and F. Hoffmann-La Roche Ltd
(Roche), has an up-front component and a running-royalty component. The
purchase price of this product includes limited, nontransferable rights under the
running-royalty component to use only this amount of the product to practice the
Polymerase Chain Reaction (PCR) and related processes described in said
patents solely for the research and development activities of the purchaser when
this product is used in conjunction with a thermal cycler whose use is covered by
the up-front fee component. Rights to the up-front fee component must be
obtained by the end user in order to have a complete license. These rights under
the up-front fee component may be purchased from Perkin-Elmer or obtained by
purchasing an Authorized Thermal Cycler. No right to perform of offer
commercial services of any kind using PCR, including without limitation
reporting the results of the purchasers activities for a fee or other commercial
considerations, is hereby granted by implication or estoppel. Further information
on purchasing licenses to practice the PCR Process may be obtained by
contacting the Director of Licensing at the Perkin-Elmer Corporation, 850
Lincoln Centre Drive, Foster City, California 94404 or at Roche Molecular
Systems, Inc., 1145 Atlantic Avenue, Alameda, California 94501.
2

26

R E L A T E D

This product optimized for use in the Polymerase Chain Reaction (PCR)
covered by patents owned by Hoffman-La Roche Inc. and F. Hoffman- La Roche
Ltd (Roche). No license under these patents to use the PCR process is conveyed
expressly or by implication to the purchaser by the purchase of this product. A
license to use the PCR Process for certain research and development activities
accompanies the purchase of certain reagents from licensed supplies such as Life
Technologies, Inc. when used in conjunction with an Authorized Thermal Cycler,
or is available form The Perkin-Elmer Corporation. Further information on
purchasing licenses to practice the PCR Process may be obtained by contacting the
Director of Licensing at the Perkin-Elmer Corporation, 850 Lincoln Centre Drive,
Foster City, California 94404 or at Roche Molecular Systems, Inc., 1145 Atlantic
Avenue, Alameda, California 94501.

5
Purchase of this product conveys to the buyer only the non-transferable right to
use the product in research conducted by the buyer. The buyer cannot sell or
otherwise transfer this product to a third party. Life Technologies, Inc. reserves all
other rights, and this product may not be used in any manner other than provided
herein. In particular, without prior written agreement between the buyer and Life
Technologies, Inc., no rights are conveyed to the buyer to use the product in
manufacturing, or to resell the product or components of the product as a standalone reagent or in a kit, whether or not such stand-alone reagent is resold or such
kit is resold for use in research. If the purchaser is not willing to accept the
limitations of this Label License, Life Technologies, Inc. is willing to accept return
of the product with a full refund. For information on purchasing a license to this
product for purposes other than research, contact the Director of Licensing, 9800
Medical Center Drive, Rockville, MD 20850. Phone (301) 610-8000. Fax (301)
610-8383.

The use of uracil-N-glycosylase for carryover prevention in amplification

reactions is the subject of claims in U.S. Patents 5,035,996, 5,638,896, and foreign
equivalents owned by Life Technologies, Inc. Purchase of this product conveys
the right to use it for research purposes only in processes claimed in the
aforementioned patents. No rights to use in other applications including the
diagnosis of disease in humans, animals, or plants, under any patents owned by
Life Technologies, Inc. are conveyed by the purchase of this product.

The purchase of this product conveys to the buyer the non-transferable right to
use the product and components of the product in research conducted by the
buyer. The buyer cannot sell or otherwise transfer this product or its components
to a third party and in particular, no rights are conveyed to the buyer to use the
product or its components for commercial purposes. Commercial purposes means
any activity for which a party receives consideration and may include, but is not
limited to, (1) use of the product or its components in manufacturing, (2) use of
the product or its components to provide a service, information or data, (3) use of
the product or its components for diagnostic purposes, or (4) resell the product or
its components, whether or not such product or its components are resold for use
in research. If purchaser is not willing to accept the limitations of this limited use
statement, Life Technologies, Inc. is willing to accept return of the products with
a full refund. For information on purchasing a license to this product for purposes
other than research, contact the Director of Licensing, 9800 Medical Center
Drive, Rockville, MD 20850. Phone (301) 610-8000. Fax (301) 610-8383.

12

P R O D U C T S

C
HA
AP
P TT E
ER
R 1
12
2
CH

Purchase of this product conveys to the buyer the non-transferable right to use
the product and components of the product in research conducted by the buyer.
The buyer cannot sell or otherwise transfer this product or its components for
commercial purposes. Commercial purposes means any activity for which a party
receives consideration and may include, but is not limited to, (1) use of the
product or its components in manufacturing, (2) use of the product or its
components to provide a service, information, or data, (3) use of the product or its
components for diagnostic purposes, or (4) resell the product or its components,
whether or not such product or its components are resold for use in research. If
purchaser is not willing to accept the limitations of this limited use statement,
Life Technologies, Inc. is willing to accept return of the products with a full
refund. For information on purchasing a license to this product for purposes other
than research, contact the Director of Licensing, 9800 Medical Center Drive,
Rockville, MD 20850. Phone (301) 610-8000. Fax (301) 610-8383.

13

14
Licensed to Life Technologies, Inc. under U.S. Patent 5,338,671, 5,587,287,
and foreign equivalents for use in research only.

The AFLP technique is covered by patents or patented applications owned by

Keygene n.v. This product is sold under license from Keygene n.v. This kit may
be used for research purposes only. For use of this kit in plant breeding, contact
the Director of Licensing, Life Technologies, Inc., 9800 Medical Center Drive,
Rockville, MD 20850. Phone (301) 610-8000. Fax (301) 610-8383. The use of
this kit for any other purpose, whether commercial or noncommercial, including
but not limited to the use for clinical, diagnostic, and/or therapeutic purposes; or
for providing services to third parties, requires a license from Keygene n.v., P.O.
Box 216, 6700 AE Wageningen, The Netherlands.
17

The use of TaqMan fluorogenic probes in 5 nuclease assays is covered by U.S.

Patent Nos. 5,210,015 and 5,487,972, owned by Roche Molecular Systems, Inc.,
and by U.S. Patent No. 5,538,848, owned by PE Corporation. Purchase of the
product does not provide a license to use this patented technology. A license to
practice this technology must be obtained from PE Biosystems, 850 Lincoln
Center Drive, Forest City, CA 94404, or from Roche Molecular Systems, Inc.,
1145 Atlantic Avenue, Alameda, CA 94501.

24

Unless indicated otherwise, purchase of this product does not convey to the
purchaser a license to practice any claim of any patent, including but not limited
to U.S. Patents 5,075,217 and 5,766,847, German Patent 3,834,636, and foreign
equivalents. Users of this product should determine if any license is required
under these or any other patents.

15

Licensed to Life Technologies, Inc. under U.S. Patents 5,496,562, 5,807,527,

5,756,126, and foreign equivalents for laboratory research and human identity
testing. Purchase of this product conveys to the buyer only the non-transferable
right to use the product for laboratory research and non-commercial human
identity testing. Use of this product for any commercial purposes or alteration of
FTA Cards and related products in any manner is prohibited without the prior
written consent of Life Technologies, Inc. Material supplied by Life Technologies,
Inc. is not intended for human or animal diagnostic or therapeutic uses. For
information concerning the availability of product and licenses to practice the
patented technology for commercial purposes in human identity testing, contact
Life Technologies, Inc., Rockville, MD. For information concerning the
availability of product and licenses to practice the patented technology for clinical
diagnosis or therapeutic uses contact FITZCO, Inc., Maple Plain, MN.

16

R E L A T E D

P R O D U C T S

27

A
PP
PE
EN
ND
D II X
X A
A
AP

Selected Primer Sequences

RT-PCR Primer Sequences

Gene

Source

Primer

Sequence

-actin

Human

sense
antisense

CCTCG CCTTT GCCGA TCC

GGATC TTCAT GAGGT AGTCA GTC

0.62

70

-actinA

Rat

sense
antisense

TACAA CCTCC TTGCA GCTCC

GGATC TTCAT GAGGT AGTCA GTC

0.62

70

-actin

Mouse

sense
antisense

GTCGT ACCAC AGGCA TTGTG ATGG

GCAAT GCCTG GGTAC ATGGT GG

0.49

71,72

GAPDH

Human

sense
antisense

GGTGA AGGTC GGAGT CAACG

CAAAG TTGTC ATGGA TGACC

0.50

73,74

GAPDH

Rat

sense
antisense

GATGC TGGTG CTGAG TATGT CG

GTGGT GCAGG ATGCA TTGCT CTGA

0.20

75,76

Dynein

Rat

sense
antisense

GCGGG CGCTG GAGGA GAA

GGATC TTCAT GAGGT AGTCA GTC

12.3

18

Polymerase

Human

sense
antisense

CGCCA AATTT CTCCC CTGAA

CCGTA GTGCT GGGCA ATGTT C

6.8

18

Polymerase

Human

sense
antisense

AAGGC TGGCG GATTA CTGCC

GATGC TGCTG GTGAT GTACT C

3.5

18

Tuberous Sclerosis

Human

sense
antisense

GGAGT TTATC ATCAC CGCGG AAATA CTGAG AG

TATTT CACTG ACAGG CAATA CCGTC CAAGG

5.3

18

18S rRNA

Soybean

sense
antisense

CTTTC GATGG TAGGA TAGTG GCCT

CAATG ATCCT TCCGC AGGTT CACCT AC

1.5

Product Size (kb)

Reference

Primers do not amplify pseudogenes.

PCR Primer Sequences

Gene

Source

Primer

Sequence

HIV gag region

Viral

SK 38
SK 39

ATAAT CACTA TCCAG TAGGA GAAAT

TTTGG TCCTG TCTTA TGTCC AGAAT GC

0.11

47

-globin

Human

29923
34016

GGTGT TCCCT TGATG TAGCA CA

CCAGG ATTTT TGATG GGACA CG

4.1

47

-globin

Human

31194
34016

GCTGC TCTGT GCATC CGAGT GG

CCAGG ATTTT TGATG GGACA CG

2.8

47

28

S E L E C T E D

P R I M E R

S E Q U E N C E S

Product Size (kb)

Reference

PCR and RT-PCR Product Groupings

Plasmid DNA Isolation
CONCERT Rapid Plasmid Purification System
CONCERT High Purity Plasmid Purification System

Genomic DNA Isolation

RNA Isolation

DNAZOL Reagents
FTA Cards and Reagent
Phenol Products
PRETAQ Thermophilic Protease
Proteinase K

TRIZOL Reagents
MESSAGEMAKER System
GLASSMAX RNA System
RNASEOUT Ribonuclease Inhibitor
Proteinase K
RNA Molecular Size Standards
DEPC-treated Water

First-Strand cDNA Synthesis (RT-PCR)

SUPERSCRIPT First-Strand Synthesis RT-PCR System
SUPERSCRIPT One-Step RT-PCR Systems
THERMOSCRIPT RT-PCR Systems
PLATINUM Quantitative RT-PCR THERMOSCRIPT System
3 RACE System
5 RACE System
Custom Primers
DNAse I, Amplification Grade
dNTPs
Ribonuclease H
M-MLV RT
SUPERSCRIPT II Reverse Transcriptase
RT-PCR Primer and Control Set

PCR
Enzymes including:
Taq DNA Polymerase
PLATINUM Enzymes (high specificity)
PLATINUM Pfx DNA Polymerase (high fidelity)
PLATINUM GENOTYPE Tsp DNA Polymerase
High Fidelity Enzyme Mixes
PCR x DNA Polymerases (high GC content)
SUPERMIXES
PLATINUM Quantitative PCR SUPERMIX-UDG
ELONGASE Reagents
PCR Reagent System
Custom Primers
dNTPs
Uracil DNA Glycosylase
Distilled Water, DNase-RNase-Free
ART Tips
PCR Tubes
HOTSTART Storage and Reaction Tubes
Silicone Oil
MICROMATE Labels

Cloning PCR Products

Electrophoretic Analysis of PCR Products

CONCERT Rapid PCR Purification System

TAQUENCH Cloning Enhancer
Restriction Endonucleases
T4 DNA Ligase
Competent Cells
CLONEAMP Systems
PCR Cloning System with GATEWAY Technology

25, 50, 250, and 500 bp DNA Ladders

10, 100, and 123 bp DNA Ladders
1 Kb PLUS DNA Ladder
HORIZON Electrophoresis Apparatus
SUNRISE Electrophoresis Apparatus
Models 250, 250 EX, and 125 Power Supply
Electrophoresis Reagents
Agarose
Agarose 1000
10X BLUEJUICE Buffer
TBE Buffer
TAE Buffer
Ethidium Bromide
Kodak Digital Science EDAS Systems

P C R

A N D

R T - P C R

P R O D U C T

G R O U P I N G S

29