| 1Nucleic Acid Amplification

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CONTENTS
I. Introduction 1 D. Tli DNA Polymerase 16
A. Basic PCR 1 E. Pfu DNA Polymerase 16
B. RT-PCR 2 V. Reverse Transcriptases 17
C. Long PCR 3 A. AMV Reverse Transcriptase 17
D. Quantitative End-Point PCR 4 B. M-MLV Reverse Transcriptase 17
E. Quantitative Real-Time PCR 4 C. M-MLV Reverse Transcriptase, RNase H Minus 17
F. Rapid Amplified Polymorphic DNA Analysis 6
VI. Example of a PCR Protocol 18
G. Rapid Amplification of cDNA Ends (RACE) 7
VII. Example of an RT-PCR Protocol 19
H. Differential Display PCR 7
A. Access RT-PCR Protocol 19
I. In situ PCR 8
B. ImProm-II™ Reverse Transcription System Protocol 19
J. Hot-Start PCR 8
K. High-Fidelity PCR 9 VIII.Troubleshooting PCR and RT-PCR 22
L. PCR and DNA Sequencing: Cycle Sequencing 9 IX. References 23
M. Cloning PCR Products 9
II. General Considerations for PCR Optimization 10
A. Magnesium Concentration 10
B. Buffer Considerations 11
C. Enzyme Concentration 11
D. PCR Primer Design 11
E. Template Quality 12
F. Template Quantity 12
G. Cycling Parameters 12
H. PCR Enhancers and Additives 13
I. Nucleic Acid Cross-Contamination 13
III. General Considerations for RT-PCR 14
A. Overview of the Access and AccessQuick™ RT-PCR
Systems 14
B. Template Considerations 14
C. Reverse Transcription Primer Design 14
D. Cycle Parameters 15
IV. Thermostable DNA Polymerases 15
A. Taq DNA Polymerase 15
B. Tfl DNA Polymerase 16
C. Tth DNA Polymerase 16

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I. Introduction DNA begins as the reaction temperature is raised to the
optimum for the DNA polymerase. For most thermostable
The polymerase chain reaction (PCR) is a relatively simple
DNA polymerases, this temperature is in the range of
technique that amplifies a DNA template to produce
70–74°C. The extension step lasts approximately 1–2
specific DNA fragments in vitro. Traditional methods of
minutes. The next cycle begins with a return to 94°C for
cloning a DNA sequence into a vector and replicating it in
denaturation.
a living cell often require days or weeks of work, but
amplification of DNA sequences by PCR requires only Each step of the cycle should be optimized for each template
hours. While most biochemical analyses, including nucleic and primer pair combination. If the temperature during
acid detection with radioisotopes, require the input of the annealing and extension steps are similar, these two
significant amounts of biological material, the PCR process steps can be combined into a single step in which both
requires very little. Thus, PCR can achieve more sensitive primer annealing and extension take place. After 20–40
detection and higher levels of amplification of specific cycles, the amplified product may then be analyzed for
sequences in less time than previously used methods. These size, quantity, sequence, etc., or used in further
features make the technique extremely useful, not only in experimental procedures.
basic research, but also in commercial uses, including An animated presentation (www.promega.com
genetic identity testing, forensics, industrial quality control /paguide/animation/selector.htm?coreName=pcr01)
and in vitro diagnostics. Basic PCR has become illustrating the PCR process is available.
commonplace in many molecular biology labs where it is
used to amplify DNA fragments and detect DNA or RNA Additional Resources for Basic PCR
sequences within a cell or environment. However, PCR has
Technical Bulletins and Manuals
evolved far beyond simple amplification and detection,
and many extensions of the original PCR method have been TB254 PCR Core Systems Technical Bulletin
described. This chapter provides an overview of different (www.promega.com/tbs/tb254/tb254.html)
types of PCR methods, applications and optimization. A 9PIM750 PCR Master Mix Promega Product
detailed treatment of these methods is beyond the scope Information
of this publication. However, an extensive bibliography is (www.promega.com
provided in the References section for researchers who /tbs/9pim750/9pim750.html)
require more comprehensive information. Promega Publications
PN083 Introducing GoTaq® DNA Polymerase:
A. Basic PCR
Improved amplification with a choice of
The PCR process was originally developed to amplify short
buffers
segments of a longer DNA molecule (Saiki et al. 1985). A
(www.promega.com
typical amplification reaction includes the target DNA, a
/pnotes/83/10492_21/10492_21.html)
thermostable DNA polymerase, two oligonucleotide
primers, deoxynucleotide triphosphates (dNTPs), reaction PN078 Performance advantages designed into
buffer and magnesium. Once assembled, the reaction is Promega's PCR Master Mix
placed in a thermal cycler, an instrument that subjects the (www.promega.com
reaction to a series of different temperatures for varying /pnotes/78/9186_09/9186_09.html)
amounts of time. This series of temperature and time PN062 PCR Core Systems: Complete reagent
adjustments is referred to as one cycle of amplification. systems for DNA amplification
Each PCR cycle theoretically doubles the amount of targeted (www.promega.com
sequence (amplicon) in the reaction. Ten cycles theoretically /pnotes/62/7807_05/promega.html)
multiply the amplicon by a factor of about one thousand; More (www.promega.com/pnotes/apps/pcr/)
20 cycles, by a factor of more than a million in a matter of publications
hours. Citations
Each cycle of PCR includes steps for template denaturation, Cousin, W. et al. (2004) Cloning of hOST-PTP: The only
primer annealing and primer extension (Figure 1.1). The example of a protein-tyrosine-phosphatase the function of
initial step denatures the target DNA by heating it to 94°C which has been lost between rodent and human. Biochem.
or higher for 15 seconds to 2 minutes. In the denaturation Biophys. Res. Commun. 321, 259–65.
process, the two intertwined strands of DNA separate from Researchers used GoTaq® DNA polymerase to amplify
one another, producing the necessary single-stranded DNA 139bp and 815bp regions of hOST-PTP cDNA for detection
template for replication by the thermostable DNA and probe synthesis. The full-length 4006bp cDNA was
polymerase. In the next step of a cycle, the temperature is amplified with Pfu DNA Polymerase.
reduced to approximately 40–60°C. At this temperature, PubMed Number: 15358244
the oligonucleotide primers can form stable associations
(anneal) with the denatured target DNA and serve as
primers for the DNA polymerase. This step lasts
approximately 15–60 seconds. Finally, the synthesis of new

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Target Region

Unamplified DNA 5′
3′
3′
5′
Cycle 1
5′ 3′
Denature and
anneal primers 3′ 5′
Extend primers
5′ 3′
3′ 5′
5′ 3′
3′ 5′
Cycle 2
5′ 3′

Denature and 3′ 5′

anneal primers 5′ 3′
3′ 5′
Extend primers
5′ 3′
3′ 5′
5′ 3′
3′ 5′
5′ 3′
3′ 5′
5′ 3′
3′ 5′
Cycle 3
5′ 3′
3′ 5′

5′ 3′

Denature and 3′
anneal primers 5′ 3′
3′ 5′

5′ 3′
3′ 5′
Extend primers
5′ 3′
3′ 5′
5′ 3′
3′ 5′
5′ 3′ =Short "target" product
3′ 5′
5′ 3′
3′ 5′
5′ 3′
3′ 5′
5′ 3′
3′ 5′
5′ 3′
3′ 5′
5′ 3′ =Long product
3′ 5′
Cycles 4-30

Amplification of short "target" product

Figure 1.1. Schematic diagram of the PCR process.

B. RT-PCR or a sequence-specific primer. Alternatively, some

The thermostable DNA polymerases used in the basic PCR thermostable DNA polymerases (e.g., Tth DNA polymerase)
process require a DNA template, and as such, the technique possess a reverse transcriptase activity, which can be
is limited to the analysis of DNA samples. Yet numerous activated under certain conditions, namely using
instances exist in which the amplification of RNA would manganese instead of magnesium as a cofactor (Myers and
be preferred. This is particularly true in analyses involving Gelfand, 1991). After this initial reverse transcription step
the differential expression of genes in tissues during has produced the cDNA template, basic PCR is carried out
development or the cloning of cDNAs from rare messages. to amplify the target sequence.
In order to apply PCR to the study of RNA, the RNA The quality and purity of the starting RNA template is
sample must first be reverse transcribed to cDNA to provide crucial to the success of RT-PCR. Either total RNA or
the necessary DNA template for the thermostable poly(A)+ RNA can be used as the starting template, but
polymerase (Figure 1.2). This process is called reverse both must be intact and free of contaminating genomic
transcription (RT), hence the name RT-PCR. DNA. Specific capture of poly(A)+ RNA will enrich a
targeted message so that less of the reverse transcription
Avian myeloblastosis virus (AMV) or Moloney murine reaction is needed for the subsequent amplification. The
leukemia virus (M-MLV or MuLV) reverse transcriptases efficiency of the first-strand synthesis reaction, which can
are generally used to produce a DNA copy of the RNA
template using either random primers, an oligo(dT) primer

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be related to the quality of the RNA template, will also
More (www.promega.com
significantly impact the results of the subsequent
publications /pnotes/apps/rt_cdna/)
amplification.
Online Tools
First Strand Synthesis: Access RT-PCR System FAQ (www.promega.com
Random primer
/faq/rtpcr.html)
mRNA Citations
5′ AAAAAAAA 3′ first-strand
N6 N6 N6 N6 N6 N6 cDNA Pozo, F. et al. (1998) Evaluation of a commercially available
reverse transcription-PCR assay for diagnosis of enteroviral
Oligo(dT) primer infection in archival and prospectively collected
mRNA
5′ AAAAAAAA 3′ first-strand
PCR
cerebrospinal fluid specimens. J. Clin. Microbiol. 36, 1741–5.
3′ T T T T T T T T 5′ cDNA
The authors compared a commercially available
enterovirus-specific RT-PCR kit with a kit of their own
Sequence-specific primer ( )
mRNA design. Their 'in-house' RT-PCR system used the Access
5′ AAAAAAAA 3′

1439MA04_6A
first-strand
3′ 5′ cDNA
RT-PCR System to amplify enterovirus sequences from
cerebral spinal fluid (CSF) extracts followed by nested PCR
Figure 1.2. Schematic diagram of RT-PCR. for final amplification of the target. Both the commercial
system and the 'in-house' system were adequate for
Additional Resources for RT-PCR amplification of enterovirus sequences from CSF of infected
individuals. Other, more time-consuming assays were
Technical Bulletins and Manuals
performed to verify the results from the RT-PCR tests.
TB220 Access RT-PCR System Technical Bulletin
PubMed Number: 9620411
(www.promega.com/tbs/tb220/tb220.html)
TM236 ImProm-II™ Reverse Transcription System Capozzo, A.V. et al. (2003) Development of DNA vaccines
Technical Manual against hemolytic-uremic syndrome in a murine model.
(www.promega.com Infect. Immun. 71, 3971–8.
/tbs/tm236/tm236.html) Researchers used the pGEM®-T Vector System to clone the
TB099 Reverse Transcription System Technical entire 1.4kb Shiga toxin type 2 gene (Stx2) from E. coli
Bulletin O157-H7 C600 (933W). The resultant construct, named
(www.promega.com/tbs/tb099/tb099.html) pGEMTStx2, was used as a template in PCR to amplify
9PIA170 AccessQuick™ RT-PCR System Promega each region of the gene corresponding to Shiga toxin type
Product Information 2 subunits A and B. Each PCR product was digested with
(www.promega.com BamH I and EcoR I, then ligated into pCDNA 3.1+ to create
/tbs/9pia170/9pia170.html) pStx2ΔA and pStx2B. Mice were then immunized with
Promega Publications either one or both of these constructs and another construct
expressing murine granulocyte-macrophage
PN079 AccessQuick™ RT-PCR System: Simple,
colony-stimulating factor. Expression of each subunit in
stable and sensitive
mouse tissue was verified by RT-PCR using specific primers
(www.promega.com
and the AccessQuick™ RT-PCR System.
/pnotes/79/9492_12/9492_12.html)
PubMed Number: 12819084
PN079 Using ImProm-II™ Reverse Transcription
System for coupled RT-PCR
(www.promega.com C. Long PCR
/pnotes/79/9492_15/9492_15.html) Amplification of long DNA fragments is desirable for
PN078 Technically speaking: Promega RT-PCR numerous applications such as physical mapping
systems explained applications (Rose, 1991) and direct cloning from genomes.
(www.promega.com While basic PCR works well when smaller fragments are
/pnotes/78/9186_21/9186_21.html) amplified, the efficiency of amplification (and therefore the
yield of amplified fragments) decreases significantly as the
PN073 Using the Access RT-PCR System:
size of the amplicon increases over 5kb. This decrease in
Reaction parameters that affect efficient
yield is attributable to the accumulation of truncated
amplification
products, which are not suitable substrates for the
(www.promega.com
subsequent cycles of amplification. This is evidenced by
/pnotes/73/8235_14/8235_14.html)
the presence of smeared, as opposed to discrete, bands on
PN071 Technically speaking: Reverse a gel.
transcription and amplification
(www.promega.com In 1994, Wayne Barnes (Barnes, 1994) and other researchers
/pnotes/71/7807_22/7807_22.html) (Cheng et al. 1994) analyzed the factors affecting
polymerization across larger regions of DNA by
thermostable DNA polymerases and identified key

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variables affecting the yield of longer PCR fragments. Most quantitative PCR uses fewer amplification cycles than basic
significant of these was an approach relying on a mixture PCR. This can cause problems in the detection of the final
of two thermostable polymerases. The first polymerase product because there is less product to detect.
lacks a 3′→5′ exonuclease (proofreading) activity; the second To monitor the efficiency of amplification, many
enzyme, present at a reduced concentration, contains a applications are designed to include an internal standard
potent proofreading activity. Presumably, when the in the PCR. One such approach includes a second primer
nonproofreading DNA polymerase (e.g., Taq DNA pair that is specific for a “housekeeping” gene (i.e., a gene
polymerase) misincorporates a dNTP, subsequent extension that has constant expression levels among the samples
of the newly synthesized DNA either proceeds very slowly compared) in the reaction (Gaudette and Crain, 1991;
or stops completely. The proofreading polymerase (e.g., Murphy et al. 1990). Amplification of housekeeping genes
Pfu DNA polymerase or Tli DNA polymerase) serves to verifies that the target nucleic acid and reaction components
remove the misincorporated nucleotide, allowing the DNA were of acceptable quality but does not account for
polymerases to continue extension of the new strand. differences in amplification efficiencies due to differences
Although the use of two thermostable DNA polymerases in product size or primer annealing efficiency between the
can significantly increase yield, other conditions can have internal standard and target being quantified.
a significant impact on the yield of longer PCR products The concept of competitive PCR—a variation of quantitative
(Cheng et al. 1995). Logically, longer extension times can PCR—is a response to this limitation. In competitive PCR,
increase the yield of longer PCR products because fewer a known amount of a control template is added to the
partial products are synthesized. Extension times of 10–20 reaction. This template is amplified using the same primer
minutes are common and depend on the length of the pair as the experimental target molecule but yields a
target. In addition, template quality is crucial. Depurination distinguishable product (e.g., different size, restriction
of the template, which is promoted at elevated temperatures digest pattern, etc.). The amounts of control and test
and lower pH, will result in more partial products and product are compared after amplification. While these
decreased overall yield. In long PCR, the denaturation time approaches control for the quality of the target nucleic acid,
is reduced to 2–10 seconds to decrease depurination of the buffer components and primer annealing efficiencies, they
template. Additives, such as glycerol and dimethyl have their own limitations (Siebert and Larrick, 1993;
sulfoxide (DMSO), also help lower the strand-separation McCulloch et al. 1995), including the fact that many depend
and primer-annealing temperatures, alleviating some of on final analysis by electrophoresis.
the depurination effects of high temperatures. Cheng et al.
Numerous fluorescent solution and solid-phase assays have
(Cheng et al. 1995) also found that reducing potassium
been described to measure the amount of amplification
concentrations by 10–40% increased the efficiency of
product generated in each reaction, but they can fail to
amplification of longer products.
discriminate amplified DNA of interest from nonspecific
D. Quantitative End-Point PCR amplification products. Some of these analyses rely on
PCR and RT-PCR are generally used in a qualitative format blotting techniques, which introduce another variable due
for evaluating biological samples. However, a wide variety to nucleic acid transfer efficiencies, while other assays have
of applications, such as the determining viral load, been developed to eliminate the need for gel electrophoresis
measuring responses to therapeutic agents and yet provide the requisite specificity. Real-time PCR, which
characterizing gene expression, would be improved by provides the ability to view the results of each amplification
quantitative determination of target abundance. cycle, is a popular way of overcoming the need for analysis
Theoretically, this should be easy to achieve, given the by electrophoresis.
exponential nature of PCR, because a linear relationship
exists between the number of amplification cycles and the Additional Resources for Quantitative PCR
logarithm of the number of molecules. In practice, however, Promega Publications
the efficiency of amplification is decreased because of PN068 Quantitative RT-PCR: Rapid construction
contaminants (inhibitors), competitive reactions, substrate of templates for competitive amplification
exhaustion, inactivation of the polymerase and target (www.promega.com
reannealing. As the number of cycles increases, the /pnotes/68/7381_16/7381_16.html)
amplification efficiency decreases, eventually resulting in
a plateau effect. E. Quantitative Real-Time PCR
Normally, quantitative PCR requires the measurement to The use of fluorescently labeled oligonucleotide probes or
be taken before the plateau phase, so the relationship primers or DNA-binding fluorescent dyes, such as SYBR®
between the number of cycles and molecules is relatively Green, to detect and quantitate a PCR product allows
linear. This point must be determined empirically for quantitative PCR to be performed in real time.
different reactions because of the numerous factors that DNA-binding dyes are easy to use but do not differentiate
can affect the amplification efficiency. Because the between specific and nonspecific PCR products.
measurement is taken prior to the reaction plateau, Fluorescently labeled nucleic acid probes have the
advantage that they react with only specific PCR products.

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These probes can also be used to detect single nucleotide the 3′-end are in close proximity, resulting in little
polymorphisms (Lee et al. 1993; Bernard et al. 1998). fluorescent signal. The hairpin probe is designed so that
However in many cases, these approaches are not the probe binds preferentially to the target DNA rather
conducive to multiplex reactions, and there is no convenient than retains the hairpin structure. As the reaction
way of distinguishing specific and nonspecific PCR progresses, increasing amounts of the probe anneal to the
products. A more recent technology, the Plexor™ accumulating PCR product, and as a result, the fluor and
technology, requires only a single fluorescently labeled quencher become physically separated. The fluor is no
primer, is compatible with multiplex PCR and allows longer quenched, and the level of fluorescence increases.
specific and nonspecific amplification products to be One advantage of this technique is that hairpin probes are
differentiated (Sherrill et al. 2004; Frackman et al. 2006). less likely to mismatch than hydrolysis probes (Tyagi et al.
Real-time PCR using labeled oligonucleotide probes or 1998). However, preliminary experiments must be
primers employs two different fluorescent reporters and performed to show that the signal is specific for the desired
relies on the transfer of energy from one reporter (the PCR product and that nonspecific amplification does not
energy donor) to the second reporter (the energy acceptor) occur.
when the reporters are in close proximity. The second The use of simple hybridization probes involves using two
reporter can be a quencher or a fluor. If the second reporter labeled probes or using one labeled probe and a labeled
is a quencher, the energy from the first reporter is absorbed PCR primer. In the first approach, the energy emitted by
but re-emitted as heat rather than light, leading to a the fluor on one probe is absorbed by a fluor on the second
decrease in the fluorescent signal. Alternatively, if the probe, which hybridizes nearby. In the second approach,
second reporter is a fluor, the energy can be absorbed and the emitted energy is absorbed by a second fluor that has
re-emitted at another wavelength through fluorescent been incorporated into the PCR product as part of the PCR
resonance energy transfer (FRET, reviewed in Didenko, primer. Both of these approaches result in increased
2001), and the progress of the reaction can be monitored fluorescence of the energy acceptor and decreased
by the decrease in fluorescence of the energy donor or the fluorescence of the energy donor. The use of hybridization
increase in fluorescence of the energy acceptor. During the probes can be simplified even further so that only one
exponential phase of PCR, the change in fluorescence is labeled probe is required. In this approach, quenching of
proportional to the accumulation of PCR product. To the fluor by deoxyguanosine is used to bring about a change
simplify quantitation, specially designed instruments in fluorescence (Crockett and Wittwer, 2001; Kurata et al.
perform both the thermal cycling steps to amplify the target 2001). The labeled probe anneals so that the fluor is in close
and the fluorescence detection to monitor the change in proximity to G residues within the target sequence, and as
fluorescence in real time during each PCR cycle. probe annealing increases, the level of fluorescence in the
There are several general categories of real-time PCR reaction decreases due to deoxyguanosine quenching. With
probes, including hydrolysis, hairpin and simple this approach, the location of probe is limited because the
hybridization probes. These probes contain a probe must hybridize so the fluorescent dye is very near a
complementary sequence that allows the probe to anneal G residue. The advantage of simple hybridization probes
to the accumulating PCR product, but probes can differ in is their ability to be multiplexed more easily than hydrolysis
the number and location of the fluorescent reporters. and hairpin probes through the use of differently colored
fluors and probes with different melting temperatures
Hydrolysis probes are labeled with a fluor at the 5′-end (reviewed in Wittwer et al. 2001).
and a quencher at the 3′-end, and because the two reporters
are in close proximity, the fluorescent signal is quenched. The Plexor™ qPCR and qRT-PCR Systems require no
During the annealing step, the probe hybridizes to the PCR probes, only two PCR primers, one of which is fluorescently
product generated in previous amplification cycles. The labeled. These systems take advantage of the specific
interaction between two modified nucleotides (Sherrill et
resulting probe:target hybrid is a substrate for the 5′→3′ al. 2004; Johnson et al. 2004; Moser and Prudent, 2003). The
exonuclease activity of the DNA polymerase, which two novel bases, isoguanine (iso-dG) and
degrades the annealed probe and liberates the fluor 5′-methylisocytosine (iso-dC), form a unique base pair in
(Holland et al. 1991). The fluor is freed from the effects of double-stranded DNA ( Johnson et al. 2004). To perform
the energy-absorbing quencher, and the progress of the fluorescent quantitative PCR using this new technology,
reaction and accumulation of PCR product is monitored one primer is synthesized with an iso-dC residue as the
by the resulting increase in fluorescence. With this 5′-terminal nucleotide and a fluorescent label at the 5′-end;
approach, preliminary experiments must be performed the second primer is unlabeled. During PCR, this labeled
prior to the quantitation experiments to show that the signal primer is annealed and extended, becoming part of the
generated is proportional to the amount of the desired PCR template used during subsequent rounds of amplification,
product and that nonspecific amplification does not occur. and the complementary iso-dGTP, which is available in the
Hairpin probes, also known as molecular beacons, contain nucleotide mix as dabcyl-iso-dGTP, pairs specifically with
inverted repeats separated by a sequence complementary iso-dC. When the dabcyl-iso-dGTP is incorporated, the
to the target DNA. The repeats anneal to form a hairpin close proximity of dabcyl and the fluorescent label on the
structure, where the fluor at the 5′-end and a quencher at opposite strand effectively quenches the fluorescent signal.

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This process is illustrated in Figure 1.3. The initial
Additional Resources for Real-Time PCR
fluorescence level of the labeled primers is high in Plexor™
System reactions. As amplification product accumulates, Technical Bulletins and Manuals
the signal decreases. TM262 Plexor™ qPCR System Technical Manual
(www.promega.com
Fluorescent
iso-dC Taq /tbs/tm262/tm262.html)
Reporter
TM263 Plexor™ One-Step qRT-PCR System
Primer Annealing Technical Manual
and Extension (www.promega.com
Taq /tbs/tm263/tm263.html)
TM264 Plexor™ Two-Step qRT-PCR System
Technical Manual
Incorporation of (www.promega.com
iso-dGTP Dabcyl-iso-dGTP /tbs/tm264/tm264.html)
Dabcyl Promega Publications
PN092 The Plexor™ Systems provide accurate
quantitation in multiplex qPCR and
qRT-PCR
Fluorescence
4909MA

(www.promega.com
Quenching
/pnotes/92/13408_10/13408_10.html)
Figure 1.3. Quenching of the fluorescent signal by dabcyl during
product accumulation.
PN090 Plexor™ technology: A new chemistry for
real-time PCR
Quenching of the fluorescent label by dabcyl is a reversible (www.promega.com
process. Fluorescence is quenched when the product is /pnotes/90/12727_02/12727_02.html)
double-stranded. Denaturing the product separates the
label and quencher, resulting in an increased fluorescent F. Rapid Amplified Polymorphic DNA Analysis
signal. Consequently, thermal melt curves can be generated Genetic analysis of organisms (animals, plants and bacteria)
by allowing all product to form double-stranded DNA at at the molecular level is an important and widely practiced
a lower temperature (approximately 60°C) and slowly area of genetic science. Several techniques developed over
ramping the temperature to denaturing levels more than a decade offer the opportunity to identify each
(approximately 95°C). The product length and sequence individual or type of individual in a species uniquely and
impact melting temperature (Tm), so the melt curve is used unambiguously. PCR has impacted this area of analysis
to characterize amplicon homogeneity. Nonspecific because of its ease of use and simplicity over traditional
amplification can be identified by broad peaks in the melt VNTR- and RFLP-based methods (Jeffreys et al. 1985;
curve or peaks with different Tm values. By distinguishing Sambrook and Russell, 2001).
specific and nonspecific amplification products, the melt One important PCR-based genetic analysis is random
curve adds a quality control aspect during routine use. The amplified polymorphic DNA analysis (RAPD; reviewed in
generation of melting curves is not possible with McClelland and Welsh, 1994; Power, 1996; Black, 1993).
technologies that rely on the 5′→3′ exonuclease activity of RAPD uses small, nonspecific primers for the amplification
Taq DNA polymerase. of regions of genomic DNA. Successful primer pairs
produce different banding profiles of PCR products
A benefit of the Plexor™ technology over detection using between individuals, strains, cultivars or species when
simple DNA-binding dyes, such as SYBR® Green, is the analyzed by gel electrophoresis.
capacity for multiplexing. The labeled primer can be tagged
Slight modifications to the basic PCR method are made for
with one of many common fluorescent labels, allowing
RAPD. First, the primers are approximately 10 bases in
two- to four-color multiplexing, depending on the
length compared to the 17- to 23-base primer length of
instrument used. The simplicity of primer design for the
normal PCR. Because the primers are shorter, the
Plexor™ technology is a distinct advantage over
temperature of the annealing reaction is reduced to less
probe-based quantitative PCR approaches. Also the
than 40°C.
Plexor™ technology does not rely on enzymatic cleavage
to generate signal and does not have the complex As with most PCR techniques, RAPD requires very little
hybridization kinetics that can be typical of other material for analysis and is relatively insensitive to the
approaches to real-time PCR. The Plexor™ technology can integrity of the material. No blotting techniques are
also be used for quantitative RT-PCR. required, thus eliminating the use of 32P, bypassing probe
generation and decreasing the amount of time required to
obtain results.

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G. Rapid Amplification of cDNA Ends (RACE) 5′ AAAAAAAAA 3′ mRNA

Rapid amplification of cDNA ends (RACE) is a variation sequence-specific primer ( )

reverse transcriptase
of RT-PCR that amplifies unknown cDNA sequences
5′ AAAAAAAAA 3′ mRNA
corresponding to the 3′- or 5′-end of the RNA. Numerous 3′ 5′ cDNA
variations of the original protocols have been published RNase or RNase activity
(Troutt et al. 1992; Edwards et al. 1991; Edwards et al. 1993; of reverse transcriptase
Liu and Gorovsky, 1993; Fromont-Racine et al. 1993; 3′ 5′ cDNA
reviewed in Schaefer, 1995) but will not be discussed in terminal transferase (TdT)
detail here. dNTP (e.g., dCTP)

3′ CCCCCC 5′
Two general RACE strategies exist: one amplifies 5′ cDNA cDNA
ends (5′ RACE) and the other captures 3′ cDNA end Taq DNA polymerase
anchor primer
sequences (3′ RACE). In either strategy, the first step in the 5′ NNNGGGGGG 3′ ds cDNA
RACE reaction involves the conversion of RNA into 3′ CCCCCC 5′
single-stranded cDNA using a reverse transcriptase. For
the subsequent amplification reaction, two PCR primers
are designed to flank the unknown sequence. One PCR PCR

primer is complementary to known sequences within the

gene, and a second primer is complementary to an “anchor” 5′ AAAAAAAAA 3′ mRNA
site (anchor primer). The anchor site may be present sequence-specific primer ( )
naturally, such as the poly(A) tail of most mRNAs, or can reverse transcriptase

be added in vitro after completion of the reverse 5′ AAAAAAAAA 3′ mRNA

3′ 5′
transcription step. The anchor primer can also carry adaptor cDNA
RNase or RNase activity
sequences, such as restriction enzyme recognition sites, to of reverse transcriptase
facilitate subsequent cloning of the amplified product. 3′ 5′ cDNA
Amplification using these two PCR primers results in a
T4 RNA ligase
product that spans the unknown 5′ or 3′ cDNA sequence, anchor primer ( )
and sequencing this product will reveal the unknown 3′ 5′ cDNA
sequence. The information obtained from partial cDNA
Taq DNA polymerase
sequences can then be used to assemble the sequence of sequence-specific primer
the full-length cDNA (Frohman et al. 1988; Loh et al. 1989; complement to anchor primer ( )

Ohara et al. 1989). 3′ ds cDNA

3′ 5′
In 5′ RACE (Figure 1.4), the first-strand cDNA synthesis

1440MA04_6A
reaction is primed using an oligonucleotide complementary
to a known sequence in the gene. After removing the RNA PCR
template, an anchor site at the 3′-end of the single-stranded Figure 1.4. Schematic diagram of two 5′ RACE methods.
cDNA is created using terminal deoxynucleotidyl
transferase, which adds a nucleotide tail. A typical 5′ AAAAAAAAA 3′ mRNA
amplification reaction follows using an anchor primer reverse transcriptase
anchor primer (VT T T T ) V=G, C or A
complementary to the newly added tail and another primer
complementary to a known sequence within the gene. Some 5′ AAAAAAAAA 3′ mRNA
3′ TTTT
variations to the original 5′ RACE procedure use different 5′ cDNA
RNase or RNase activity
approaches to add an anchor site adjacent to the 5′-end of reverse transcriptase
sequences of the cDNA. One of these approaches, 3′ TTTT cDNA
single-stranded ligation of cDNA (SLIC), uses RNA ligase Taq DNA polymerase
5′

to covalently join an oligonucleotide anchor site adjacent sequence-specific primer ( )

to the 3′-end of the single-stranded cDNA. anchor primer
5′ 3′ ds cDNA
The 3′-RACE procedure (Figure 1.5) uses an oligo(dT) 3′ TTTT 5′
primer/adaptor as a primer for the reverse transcription
PCR
reaction. The oligo(dT) primer anneals to the poly(A)+ tail
1441MA04_6A

of the mRNA. This oligo(dT) primer/adaptor is also used 5′ 3′

3′ 5′
as the anchor primer in the subsequent amplifications along
with a primer complementary to known sequences within Figure 1.5. Schematic diagram of a typical 3′-RACE protocol.
the gene.
H. Differential Display PCR
Differential display PCR (DDPCR) is another variation of
RT-PCR and is used to identify differences in mRNA
expression patterns between two cell lines or populations.

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In one example of this procedure, cDNA synthesis is primed polymerase is also required unless BSA is added to the
using a set of modified oligo(dT) primers, which anneal to reaction, presumably because the polymerase binds to the
the poly(A)+ tail of mRNA (Liang and Pardee, 1992). Each glass plate and coverslip.
of the oligo(dT) primers carries an additional two Tissue preparation also plays a significant role in the
nucleotides at the 3′-end. This ensures that extension only success of in situ PCR. A strong relationship exists between
occurs if the primer anneals immediately adjacent to the the time of fixation and protease digestion and the intensity
junction between the poly(A)+ tail and 3′ end of the mRNA. of PCR signal. Tissue preparation also impacts the amount
Because the two additional nucleotides will only anneal to of side reaction, resulting in primer-independent signals,
a subset of the mRNA molecules, this also reduces the which are not normally present in basic PCR. These
complexity of the RNA population that is reverse primer-independent signals often arise from Taq DNA
transcribed. The RNA is first reverse transcribed with one polymerase-mediated repair of single-stranded gaps in the
of the modified oligo(dT) primers to synthesize first-strand genomic DNA.
cDNA, which is then amplified by PCR using two random
10mer primers. After amplification, the reaction products As the use of the technique has spread, the process has been
are visualized by gel electrophoresis, and comparisons of further optimized. Numerous publications (reviewed in
banding patterns are made between the two cell Nuovo, 1995; Staskus et al. 1995) describe process
populations to identify differentially expressed cDNAs. improvements that increase sensitivity and decrease
nonspecific amplification products. Additionally, several
Another form of analyzing differences between complex thermal cycler manufacturers have introduced instruments
genomes is representational difference analysis (RDA). This designed specifically for in situ amplification.
method combines “subtractive” library techniques (Lisitsyn
et al. 1993) with PCR amplification to find differences in J. Hot-Start PCR
complex genomes. A variation of this is cDNA RDA, where Hot-start PCR is a common technique to reduce nonspecific
total RNA from the cell populations is first converted into amplification due to the assembly of amplification reactions
cDNA, subtractive techniques are performed and the at room temperature or on ice. At these lower temperatures,
products are amplified by PCR (Hubank and Schatz, 1994). PCR primers can anneal to template sequences that are not
By using cDNA, the complexity is significantly reduced, perfectly complementary. Since thermostable DNA
providing another method to analyze differences in polymerases have activity at these low temperatures
expression between cell types or in response to various (although in most cases the activity is less than 25%) the
treatments. In this regard, cDNA RDA can be used as an polymerase can extend misannealed primers. This newly
alternative to DDPCR. synthesized region is perfectly complementary to the DNA
template, allowing primer extension and the synthesis of
Additional Resources for Differential Display PCR undesired amplification products. However, if the reaction
Promega Publications is heated to temperatures >60°C before polymerization
NN015 Targeted display: Identifying differentially begins, the stringency of primer annealing is increased, and
expressed mRNAs the subsequent synthesis of undesired PCR products is
(www.promega.com avoided or reduced.
/nnotes/nn502/502_13.htm) Hot-start PCR can also reduce the amount of primer-dimer
synthesized by increasing the stringency of primer
I. In situ PCR annealing. At lower temperatures, the PCR primers can
In situ PCR, first described in 1990, combines the sensitivity anneal to each other via regions of complementarity, and
of PCR or RT-PCR amplification with the cellular or the DNA polymerase can extend the annealed primers to
histological localization associated with in situ produce primer dimer, which often appears as a diffuse
hybridization techniques (Haase et al. 1990). These features band of approximately 50–100bp on an ethidium
make in situ PCR a powerful tool for detecting proviral bromide-stained gel. The formation of nonspecific products
DNA, oncogenesis and localization of rare messages. and primer-dimer can compete for reagent availability with
The technique is amenable to analysis of fixed cells or tissue the amplification of the desired product. Thus, hot-start
cross-sections. Detection of amplified products can be PCR can improve the yield of the specific PCR products.
accomplished indirectly by subsequent hybridization using To perform hot-start PCR, the reactions are assembled on
either radiolabeled, fluorescently labeled or biotin-labeled ice or at room temperature, but one critical component is
nucleic acid probes. PCR products can also be detected omitted until the reaction has been heated to 60–65°C, at
directly by the incorporation of a labeled nucleotide, which point the missing reagent is added. This omission
although this method is subject to higher background levels. prevents the polymerase from extending primers until the
The use of in situ PCR requires altering some of the reaction the critical component is added at the higher temperature
parameters typical of basic PCR (Nuovo et al. 1993; Thaker, where primer annealing is more stringent. However, this
method is tedious and increases the risk of contamination.
1999). For example, increased Mg2+ concentrations
A second, less labor-intensive approach involves the
(approximately 4.5mM versus the normal 1.5–2.5mM) are
reversible inactivation or physical separation of one or more
used for in situ PCR. An increased amount of DNA

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critical components in the reaction. For example, the higher fidelity at higher pH (Cline, 1996). Finally, exposing
magnesium or DNA polymerase can be sequestered in a the DNA template to very high temperatures (i.e., 94°C)
wax bead, which melts as the reaction is heated to 94°C for extended periods of time can lead to DNA damage,
during the denaturation step, releasing the component only specifically the release of bases from the phosphodiester
at higher temperatures (Carothers et al. 1989; Krishnan et backbone. The resulting abasic sites can cause some DNA
al. 1991; Clark, 1988). Alternatively, the DNA polymerase polymerases to stall but can also result in a higher rate of
can be kept in an inactive state by binding to an mutations, most frequently transversions, as the DNA
oligonucleotide, also known as an aptamer (Lin and polymerase adds a random nucleotide at an abasic site
Jayasena, 1997; Dang and Jayasena, 1996) or an antibody (Eckert and Kunkel, 1991).
(Scalice et al. 1994; Sharkey et al. 1994). This bond is
disrupted at the higher temperatures, releasing the Additional Resources for High-Fidelity PCR
functional DNA polymerase. Promega Publications
PN068 Pfu DNA Polymerase: A high fidelity
Additional Resources for Hot-Start PCR
enzyme for nucleic acid amplification
Technical Bulletins and Manuals (www.promega.com
TB247 TaqBead™ Hot Start Polymerase Technical /pnotes/68/7381_07/7381_07.html)
Bulletin
(www.promega.com/tbs/tb247/tb247.html) L. PCR and DNA Sequencing: Cycle Sequencing
Promega Publications The PCR process has also been applied to DNA sequencing
PN060 Improved PCR amplification using in a technique called cycle sequencing (Murray, 1989; Saluz
TaqBead™ Hot Start Polymerase and Jost, 1989; Carothers et al. 1989; Krishnan et al. 1991).
(www.promega.com The main differences between a cycle sequencing reaction
/pnotes/60/6079_02/promega.html) and a typical DNA sequencing reaction is the choice of
polymerase and the incorporation of thermal cycling. Cycle
K. High-Fidelity PCR sequencing reactions use thermostable polymerases, while
For some applications, such as gene expression, the conventional reactions use thermolabile polymerases,
mutagenesis or cloning, the number of mutations such as modified T7 DNA polymerase or the Klenow
introduced during PCR needs to be minimized. For these fragment of E. coli DNA polymerase I.
applications, we recommend using a proofreading Cycle sequencing reactions also differ from typical PCR
polymerase. Proofreading DNA polymerases, such as Pfu amplification reactions in that they use only a single primer,
and Tli DNA polymerases, have a 3′→5′ exonuclease resulting in a linear (as opposed to theoretically
activity, which can remove any misincorporated exponential) amplification of the target molecule. Other
nucleotides, and so the error rate is relatively low. The reaction components are comparable, and either radioactive
accuracy of Pfu DNA polymerase is approximately twofold or fluorescent labels may be incorporated for detection.
higher than that of Tli DNA polymerase and 6-fold higher
than that of Taq DNA polymerase (Cline, 1996). Additional Resources for Cycle Sequencing
The most commonly used DNA polymerase for PCR is Taq Technical Bulletins and Manuals
DNA polymerase, which has an error rate of approximately TM024 fmol® DNA Cycle Sequencing System
1 × 10–5 errors per base. This error rate is relatively high (www.promega.com
due to the enzyme's lack of 3′→5′ exonuclease /tbs/tm024/tm024.html)
(proofreading) activity. The error rate of Tfl DNA Promega Publications
polymerase, another nonproofreading polymerase, is PN044 Rapid PCR sequencing of plasmid DNA
similar to that of Taq DNA polymerase. directly from colonies of Saccharomyces
cerevisiae.
Reaction conditions can affect DNA polymerase fidelity,
(www.promega.com
and DNA polymerases may be affected in different ways
/pnotes/44/rapid/rapid.html)
or to different degrees. In general, excess magnesium or
the presence of manganese will cause the fidelity of DNA PN040 Direct sequencing of PCR products with
polymerases to be reduced (Eckert and Kunkel, 1990). degenerate primers
Unequal nucleotide concentrations can also affect fidelity; (www.promega.com
nucleotides that are present at higher concentrations will /pnotes/40/fmol2/fmol2.htm)
be misincorporated at a higher frequency (Eckert and
M. Cloning PCR Products
Kunkel, 1990). Reaction pH can also have a big effect on
fidelity (Eckert and Kunkel, 1990; Eckert and Kunkel, 1991). Amplification with a DNA polymerase lacking 3′→5′
For example, the fidelity of Taq DNA polymerase increases (proofreading) exonuclease activity (e.g., Taq DNA
as pH decreases, with the lowest error rate occurring in the polymerase) yields products that contain a single
range of pH 5–6 (Eckert and Kunkel, 1990), but the opposite 3′-terminal nucleotide overhang, typically an A residue
is true for Pfu DNA polymerase. Pfu DNA polymerase has (Clark, 1988; Hu, 1993). Before this overhang was identified,

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blunt-end cloning of PCR products was inefficient at best.
Citations
However, these PCR products can now be conveniently
cloned into T-vectors, which contain a single T overhang Chun, T. et al. (1999) Molecular cloning and characterization
(reviewed in Mezei and Storts, 1994; Guo and Bi, 2002). of a novel CD1 gene from the pig. J. Immunol. 162, 6562–71.
The complete cDNA for the CD1.1 gene was amplified and
DNA polymerases that possess proofreading activity (e.g.,
subcloned into the pTARGET™ Mammalian Expression
Tli DNA polymerase or Pfu DNA polymerase) generate
Vector. The 339 amino acid protein was stably expressed
blunt-ended PCR products. These products are compatible
in CHO cells following selection with G-418 sulfate.
with standard blunt-end cloning strategies. Conversely,
Expression was confirmed by Northern blot and FACS®
blunt-end PCR products can be tailed with Taq DNA
polymerase and dATP prior to cloning into a T-vector analysis with a mAb to the CD1.1 protein. The pGEM®-T
(Zhou et al. 1995). Vector System was used for routine cloning of both PCR
and RT-PCR products.
Additional Resources for Cloning PCR Products PubMed Number: 10352272
Technical Bulletins and Manuals Bufler, P. et al. (2002) A complex of the IL-1 homologue
TM042 pGEM®-T and pGEM®-T Easy Vector IL-1F7b and IL-18-binding protein reduces IL-18 activity.
Systems Technical Manual Proc. Natl. Acad. Sci. USA 99, 13723–8.
(www.promega.com The cDNA for the IL-1F7b protein was amplified from a
/tbs/tm042/tm042.html) human spleen cDNA library and directly subcloned in the
TM044 pTARGET™ Mammalian Expression Vector pGEM®-T Easy Vector and sequenced. The clone was
System Technical Manual transferred to a bacterial expression vector with a
(www.promega.com purification tag. The expressed protein was used to make
/tbs/tm044/tm044.html) a polyclonal rabbit antibody and antibody purification
Promega Publications column. The cDNA for the IL-1F7b was reamplified and
PN082 Technically speaking: T-vector cloning directly subcloned into the pTARGET™ Mammalian
(www.promega.com Expression Vector. The cDNA was stably expressed in the
/pnotes/82/10203_24/10203_24.html) RAW264.7 mouse monocyte/macrophage cell line. Lysates
from the cells stably expressing IL-1F7b were used to test
PN071 Rapid ligation for the pGEM®-T and the antibodies by Western blotting. Expressing cells were
pGEM®-T Easy Vector Systems also tested by immunocytochemistry.
(www.promega.com
PubMed Number: 12381835
/pnotes/71/7807_08/7807_08.html)
PN068 Technically speaking: Optimized cloning
with T vectors II. General Considerations for PCR Optimization
(www.promega.com This discussion focuses on the use of Taq DNA polymerase
/pnotes/68/7381_31/7381_31.html) in PCR, since this is the enzyme most commonly used in
PN060 Digestion of PCR and RT-PCR products PCR. Many of these suggestions also apply when using
with restriction endonucleases without other DNA polymerases.
prior purification or precipitation A. Magnesium Concentration
(www.promega.com
Magnesium is a required cofactor for thermostable DNA
/pnotes/60/6079_23/promega.html)
polymerases, and magnesium concentration is a crucial
More (www.promega.com factor that can affect the success of the amplification.
publications /pnotes/apps/cloning/) Template DNA concentration, chelating agents present in
Vector Maps the sample (e.g., EDTA or citrate), dNTP concentration and
pGEM®-T and pGEM®-T Easy Vectors (www.promega.com the presence of proteins can all affect the amount of free
/vectors/t_vectors.htm#b01) magnesium in the reaction. In the absence of adequate free
pTARGET™ Mammalian Expression Vector magnesium, Taq DNA polymerase is inactive (Figure 1.6).
(www.promega.com/vectors/t_vectors.htm#b02) Excess free magnesium reduces enzyme fidelity (Eckert
Online Tools and Kunkel, 1990) and may increase the level of nonspecific
pGEM®-T and pGEM®-T Easy Vector Systems FAQ amplification (Williams, 1989; Ellsworth et al. 1993). For
(www.promega.com/faq/pgemt.html) these reasons, researchers should empirically determine
pTARGET™ Mammalian Expression Vector System FAQ the optimal magnesium concentration for each reaction.
(www.promega.com/faq/ptarget.html) To do so, perform a series of reactions containing
1.0–4.0mM Mg2+ in 0.5–1mM increments and visualize the
results to determine which magnesium concentration
produced the highest yield of product and the minimum
amount of nonspecific product. The effect of magnesium
concentration and the optimal concentration range can vary

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with the particular DNA polymerase. For example, the DNA Polymerase contains native Taq DNA polymerase in
performance of Pfu DNA polymerase seems to be less a proprietary formulation. It is supplied with 5X Green
dependent on magnesium concentration, but when GoTaq® Reaction Buffer and 5X Colorless GoTaq® Reaction
optimization is required, the optimal concentration is Buffer. The 5X Green GoTaq® Reaction Buffer contains two
usually in the range of 2–6mM. dyes (blue and yellow) that separate during electrophoresis
Many DNA polymerases are supplied with a to monitor migration progress. The buffer also contains a
magnesium-free reaction buffer and a tube of 25mM MgCl2, compound that increases the density of the sample, so it
will sink into the well of the agarose gel, allowing reactions
so you can adjust the Mg2+ concentration to the level that
to be directly loaded onto an agarose gel without the need
is optimal for each reaction. Before assembling the reactions,
for loading dye. The blue dye comigrates at the same rate
be sure to thaw the magnesium solution completely prior
as a 3–5kb DNA fragment in a 1% agarose gel. The yellow
to use and vortex the magnesium solution for several
dye migrates at a rate faster than primers (<50bp) in a 1%
seconds before pipetting. Magnesium chloride solutions
can form concentration gradients as a result of multiple agarose gel. The 5X Colorless GoTaq® Reaction Buffer and
freeze-thaw cycles, and vortex mixing is required to obtain the 5X Green GoTaq® Reaction Buffer have the same
a uniform solution. These two steps, though seemingly formulation, except for the dyes. The 5X Colorless GoTaq®
simple, eliminate the cause of many failed experiments. Reaction Buffer is recommended for any applications where
absorbance or fluorescence measurements will be taken of
Some scientists prefer to use reaction buffers that already
the PCR amplimer without prior clean-up. Both buffers are
contain MgCl2 at a final concentration of 1.5mM. It should
supplied at pH 8.5 and contain MgCl2 at a concentration
be noted, however, that Hu et al. reported performance
of 7.5mM for a final concentration of 1.5mM.
variability of reaction buffer solutions containing
magnesium. The free magnesium changes of 0.6mM GoTaq® Flexi DNA Polymerase is supplied with 5X Green
observed in their experiments dramatically affected GoTaq® Flexi Reaction Buffer and 5X Colorless GoTaq®
amplification yields in an allele-specific manner. The Flexi Reaction Buffer. The compositions are identical to the
authors found that heating the buffer at 90°C for 10 minutes 5X Green GoTaq® Reaction Buffer and 5X Colorless GoTaq®
restored the homogeneity of the solution. They postulated Reaction Buffer, except that the GoTaq® Flexi reaction
that magnesium chloride precipitates as a result of multiple buffers do not contain MgCl2. Instead, the GoTaq® Flexi
freeze-thaw cycles (Hu et al. 1992).
DNA Polymerase is supplied with a tube of 25mM MgCl2,
M 1 2 3 4 5 6 7 8 so reactions can be suppplemented with varying
concentrations of magnesium.
C. Enzyme Concentration
2,645 –
1,605 – We recommend using 1–1.25 units of Taq DNA polymerase
1,198 – in a 50μl amplification reaction. In most cases, this is an
excess of enzyme, and adding more enzyme will not
significantly increase product yield. In fact, increased
amounts of enzyme increase the likelihood of generating
1348TB01_6A

artifacts associated with the intrinsic 5′→3′ exonuclease

activity of Taq DNA polymerase, resulting in smeared bands
Mg2+ (mM) 0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 in an agarose gel (Longley et al. 1990; Bell and DeMarini,
Figure 1.6. Effects of magnesium concentration on PCR 1991).
amplification. PCR amplifications were performed using various Pipetting errors are a frequent cause of excessive enzyme
concentrations of Mg2+ to demonstrate this effect on the levels. Accurate dispensing of small volumes of enzyme
amplification of a 1.8kb target luciferase gene. The reaction solutions in 50% glycerol is difficult, so we strongly
products were analyzed by agarose gel electrophoresis followed
recommend preparing a reaction master mix, which
by ethidium bromide staining. Lane M, Promega pGEM® DNA
requires a larger volume of each reagent, to reduce pipetting
Markers (Cat.# G1741); Lane 1, 0mM Mg2+; Lane 2, 0.5mM Mg2+;
errors.
Lane 3, 1mM Mg2+; Lane 4, 1.5mM Mg2+; Lane 5, 2mM Mg2+; Lane
6, 2.5mM Mg2+; Lane 7, 3mM Mg2+ and Lane 8, 3.5mM Mg2+. D. PCR Primer Design
PCR primers define the target region to be amplified and
B. Buffer Considerations generally range in length from 15–30 bases. Ideally primers
Most reaction buffers consist of a buffering agent, most will have a GC-content of 40–60%. Avoid three G or C
often a Tris-based buffer, and salt, commonly KCl. The residues in a row near the 3′-end of the primer to minimize
buffer regulates the pH of the reaction, which affects the nonspecific primer annealing. Also, avoid primers with
DNA polymerase activity and fidelity. Modest intra- or intermolecular complementary sequences to
concentrations of KCl will increase DNA polymerase minimize the production of primer-dimer. Intramolecular
activity by 50–60% over activities in the absence of KCl; regions of secondary structure can interfere with primer
50mM KCl is considered optimal (Gelfand, 1989). GoTaq® annealing to the template and should be avoided.

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Ideally, the melting temperature (Tm), the temperature at concentration of the specific PCR product is often not
which 50% of the primer molecules are annealed to the known. We recommend diluting the previous amplification
complementary sequence, of the two primers will be within reaction 1:10 to 1:10,000 before reamplification.
5°C, so the primers anneal efficiently at the same 1μg of 1kb RNA = 1.77 × 1012 molecules
temperature. Primers can be designed to include sequences
that can be useful for downstream applications. For 1μg of 1kb dsDNA = 9.12 × 1011 molecules
example, restriction enzyme sites can be placed at the 1μg of pGEM® Vector DNA = 2.85 × 1011 molecules
5′-ends of the PCR primers to facilitate subsequent cloning
of the PCR product, or a T7 RNA polymerase promoter can 1μg of lambda DNA = 1.9 × 1010 molecules
be added to allow in vitro transcription without the need 1μg of E. coli genomic DNA = 2 × 108 molecules
to subclone the PCR product into a vector.
1μg of human genomic DNA = 3.04 × 105 molecules
E. Template Quality
G. Cycling Parameters
Successful amplification depends on DNA template
The two most commonly altered cycling parameters are
quantity and quality. Reagents commonly used in the
annealing temperature and extension time. The lengths
purification of nucleic acids (salts, guanidine, proteases,
and temperatures for the other steps of a PCR cycle do not
organic solvents and SDS) are potent inactivators of DNA
usually vary significantly. However in some cases, the
polymerases. For example, 0.01% SDS will inhibit Taq DNA
denaturation cycle can be shortened or a lower denaturation
polymerase by 90%, while 0.1% SDS will inhibit Taq DNA
temperature used to reduce the number of depurination
polymerase by 99.9% (Konat et al. 1994). A few other
events, which can lead to mutations in the PCR products.
examples of PCR inhibitors are phenol (Katcher and
Schwartz, 1994), heparin (Beutler et al. 1990; Holodniy et Primer sequence is a major factor that determines the
al. 1991), xylene cyanol, bromophenol blue (Hoppe et al. optimal annealing temperature, which is often within 5°C
1992), plant polysaccharides (Demeke and Adams, 1992), of the melting temperature (Tm) of the primers. Using an
and the polyamines spermine and spermidine (Ahokas and annealing temperature slightly higher than the primer Tm
Erkkila, 1993). In some cases, the inhibitor is not introduced will increase annealing stringency and can minimize
into the reaction with the nucleic acid template. A good nonspecific primer annealing, decreasing the amount of
example of this is an inhibitory substance that can be undesired products synthesized. However, using an
released from polystyrene or polypropylene upon exposure annealing temperature lower than the primer Tm can result
to ultraviolet light (Pao et al. 1993; Linquist et al. 1998).
in higher yields, as the primers anneal more efficiently at
If an amplification reaction fails and you suspect the DNA the lower temperature. We recommend testing several
template is contaminated with an inhibitor, add a control annealing temperatures, starting approximately 5°C below
DNA and primer pair that has amplified well in the past the Tm, to determine the best annealing conditions. In many
to the amplification reaction with the suspect DNA cases, nonspecific amplification and primer-dimer
preparation. Failure to amplify the control DNA usually formation can be reduced through the optimization of
indicates the presence of an inhibitor. Additional steps to annealing temperature, but if undesirable PCR products
clean up the DNA preparation, such as phenol:chloroform remain a problem, consider incorporating one of the many
extraction or ethanol precipitation, may be necessary. hot-start PCR methods.
F. Template Quantity Oligonucleotide synthesis facilities will often provide an
The amount of template required for successful estimate of the primer's Tm. The Tm can also be calculated
amplification depends upon the complexity of the DNA using the Biomath Calculators (www.promega.com
sample. For example, of a 4kb plasmid containing a 1kb /biomath/). Numerous formulas exist to determine the
target sequence, 25% of the input DNA is the target of theoretical Tm of nucleic acids (Baldino, Jr. et al. 1989;
interest. Conversely, a 1kb target sequence in the human Rychlik et al. 1990). The formula below can be used to
genome (3.3 × 109bp) represents approximately 0.00003% estimate the melting temperature for oligonucleotides:
of the input DNA. Thus, approximately 1,000,000-fold more
human genomic DNA is required to maintain the same Tm = 81.5 + 16.6 × (log10[Na+]) + 0.41 × (%G+C) – 675/n
number of target copies per reaction. Common mistakes where [Na+] is the molar salt concentration, [K+] = [Na+]
include using too much plasmid DNA, too much PCR and n = number of bases in the oligonucleotide
product or too little genomic DNA as the template.
Reactions with too little DNA template will have low yields, Example:
while reactions with too much DNA template can be To calculate the melting temperature of a 22mer
plagued by nonspecific amplification. If possible, start with oligonucleotide with 60% G+C in 50mM KCl:
>104 copies of the target sequence to obtain a signal in 25–30 Tm = 81.5 + 16.6 × (log10[0.05]) + 0.41 × (60) – 675/22
cycles, but try to keep the final DNA concentration of the
reaction ≤10ng/μl. When reamplifying a PCR product, the = 81.5 + 16.6 × (–1.30) + 24.60 – 30.68 = 54°C

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The length of the extension cycle, which may also need to In some cases, general stabilizing agents such as BSA
be optimized, depends on the size of the PCR product and (0.1mg/ml), gelatin (0.1–1.0%) and nonionic detergents
the DNA polymerase being used. In general, allow (0–0.5%) can overcome failures to amplify a region of DNA.
approximately 1 minute for every 1kb of amplicon These additives can increase DNA polymerase stability
(minimum extension time = 1 minute) for nonproofreading and reduce the loss of reagents through adsorption to the
DNA polymerases and 2 minutes for every 1kb of amplicon tube walls. BSA has also been shown to overcome the
for proofreading DNA polymerases. Avoid excessively inhibitory effects of melanin on RT-PCR (Giambernardi et
long extension times, as they can increase the likelihood of al. 1998). Nonionic detergents, such as Tween®-20, NP-40,
generating artifacts associated with the intrinsic 5′→3′ and Triton® X-100, have the added benefit of overcoming
exonuclease activity of Taq DNA polymerase (Longley et the inhibitory effects of trace amounts of strong ionic
al. 1990; Bell and DeMarini, 1991). detergents, such as 0.01% SDS (Gelfand and White, 1990).
Ammonium ions can make an amplification reaction more
PCR typically involves 25–35 cycles of amplification. The
tolerant of nonoptimal conditions. For this reason, some
risk of undesirable PCR products appearing in the reaction
PCR reagents include 10–20mM (NH4)2SO4. Other PCR
increases as the number of cycles increases, so we
recommend performing only enough cycles to synthesize enhancers include glycerol (5–20%), polyethylene glycol
the desired amount of product. If nonspecific amplification (5–15%) and tetramethyl ammonium chloride (60mM).
products accumulate before sufficient amounts of PCR I. Nucleic Acid Cross-Contamination
product can be synthesized, consider diluting the products It is important to minimize cross-contamination between
of the first reaction and performing a second amplification samples and prevent carryover of RNA and DNA from one
with the same primers or primers that anneal to sequences experiment to the next. Use separate work areas and
within the desired PCR product (nested primers). pipettors for pre- and postamplification steps. Use positive
H. PCR Enhancers and Additives displacement pipets or aerosol-resistant tips to reduce
The addition of PCR-enhancing agents can increase yield cross-contamination during pipetting. Wear gloves and
of the desired PCR product or decrease the production of change them often.
undesired products. There are many PCR enhancers, which There are a number of techniques that can be used to
can act through a number of different mechanisms. These prevent the amplification of DNA contaminants. PCR
reagents will not enhance all PCR reactions; the beneficial reagents can be treated with isopsoralen, a photo-activated,
effects are often template- and primer-specific and will cross-linking reagent that intercalates into double-stranded
need to be determined empirically. Some of the more DNA molecules and forms covalent, interstrand crosslinks,
common enhancing agents are discussed below. to prevent DNA denaturation and replication. These
The addition of betaine, DMSO and formamide can be interstrand crosslinks effectively render contaminating
helpful when amplifying GC-rich templates and templates DNA unamplifiable.
that form strong secondary structures, which can cause Treatment of the PCR reagents with uracil-N-glycosylase
DNA polymerases to stall. GC-rich templates can be (UNG), a DNA repair enzyme that hydrolyzes the
problematic due to inefficient separation of the two strands base-ribose bond at uracil residues, eliminates one of the
of DNA or the tendency for the complementary, GC-rich most common sources of DNA contamination: previously
primers to form intermolecular secondary structures, which amplified PCR products. UNG treatment prevents
will compete with primer annealing to the template. Betaine replication of uracil-containing DNA by causing the DNA
reduces the amount of energy required to separate the polymerase to stall at the resulting abasic sites. For UNG
strands of DNA templates (Rees et al. 1993). DMSO and to be an effective safeguard against contamination, the
formamide are thought to aid in amplification in a similar products of previous amplifications must have been
manner by interfering with the formation of hydrogen synthesized in the presence of dUTP. This is easily
bonds between the two strands of DNA (Geiduschek and accomplished by substituting dUTP for some or all of the
Herskovits, 1961). dTTP in the reaction. Nonproofreading polymerases will
Some reactions that amplify poorly in the absence of readily incorporate dUTP into a PCR product, but
enhancers will give a higher yield of PCR product when proofreading polymerases incorporate dUTP much less
betaine (1M), DMSO (1–10%) or formamide (1–10%) are efficiently (Slupphaug et al. 1993; Greagg et al. 1999; Lasken
added. Concentrations of DMSO greater than 10% and et al. 1996). Since the incorporation of dUTP has no
formamide greater than 5% can inhibit Taq DNA noticeable effect on the intensity of ethidium bromide
polymerase and presumably other DNA polymerases as staining or on the electrophoretic mobility of the PCR
well (Varadaraj and Skinner, 1994). Specific examples of product, the reactions can be analyzed by standard agarose
the effects of DMSO and betaine on GoTaq® DNA gel electrophoresis. While both methods are effective (Rys
Polymerase have been published in Neural Notes and Persing, 1993), UNG treatment has the advantage that
(www.promega.com/nnotes/nn021/21_02.htm)(Knoche, K., both single-stranded and double-stranded DNA templates
2002). will be rendered unamplifiable (Longo et al. 1990).

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III. General Considerations for RT-PCR pg total RNA per reaction
Please also read General Considerations for PCR bp M 106 105 104 103 102 101 1 0 M
Optimization. Many of the important parameters discussed 1,500 –
1,000 –
there also apply to RT-PCR. – 540bp
500 –
β-actin
A. Overview of the Access and AccessQuick™ RT-PCR Systems amplimer
The Access RT-PCR System and AccessQuick™ RT-PCR
System are designed for the reverse transcription and 100 –
amplification of a specific target RNA from either total

1166TD09_6A
RNA or mRNA (Miller and Storts, 1995; Knoche and
Denhart, 2001). These one-tube, two-enzyme systems
provide sensitive, quick and reproducible analysis of even
rare RNAs (Miller and Storts, 1996). The systems use AMV Figure 1.7. Amplification of a specific message in total RNA.
RT-PCR amplifications containing the indicated amounts of mouse
Reverse Transcriptase for first-strand cDNA synthesis and
liver total RNA were performed using the Access RT-PCR System
the thermostable Tfl DNA Polymerase from Thermus flavus as described in the Access RT-PCR Protocol using oligonucleotide
(Kaledin et al. 1981) for second-strand cDNA synthesis and primers specific to the mouse β-actin transcript. The specific 540bp
DNA amplification. The systems include an optimized amplicon is indicated. Equivalent aliquots of each amplification
single-buffer system that permits extremely sensitive reaction were separated on a 3% NuSieve®/ 1% agarose gel in 1X
detection of RNA transcripts without the need for buffer TAE buffer containing 0.5μg/ml ethidium bromide. Lanes M, 100bp
additions between the reverse transcription and PCR DNA Ladder (Cat.# G2101).
amplification steps. This simplifies the procedure and
reduces the potential for contaminating the samples. The C. Reverse Transcription Primer Design
improved performance of AMV reverse transcriptase at Selection of an appropriate primer for reverse transcription
elevated temperatures (45°C) minimizes problems depends on target mRNA size and the presence of
encountered with secondary structures in RNA (Brooks et secondary structure. For example, a primer that anneals
al. 1995). specifically to the 3′-end of the transcript (a
B. Template Considerations sequence-specific primer or oligo(dT) primer) may be
problematic when reverse transcribing the 5′-ends of long
For RT-PCR, successful reverse transcription depends on mRNAs or molecules that have significant secondary
the integrity and purity of the RNA used as a template. structure, which can cause the reverse transcriptase to stall
Procedures for creating and maintaining an RNase-free during cDNA synthesis. Random hexamers prime reverse
environment are described in Blumberg, 1987. The use of transcription at multiple points along the transcript. For
an RNase inhibitor (e.g., Recombinant RNasin® this reason, they are useful for either long mRNAs or
Ribonuclease Inhibitor) is strongly recommended. For transcripts with significant secondary structure.
optimal results, the RNA template, whether a total RNA
preparation, an mRNA population or a synthesized RNA Whenever possible, we recommend using a primer that
transcript, should be DNA-free. Tfl DNA Polymerase anneals only to defined sequences in particular RNAs
(supplied with the Access and AccessQuick™ RT-PCR (sequence-specific primers) rather than to the entire RNA
Systems) has no reverse transcriptase activity under the population in the sample (e.g., random hexamers or
standard reaction conditions (Miller and Storts, 1995), but oligo(dT) primer). To differentiate between amplification
amplification products will be generated if the template of cDNA and amplification of contaminating genomic DNA,
contains trace amounts of DNA with similar sequences. design primers to anneal to sequences in exons on opposite
Excellent amplification results can be obtained with the sides of an intron, so any amplification product derived
Access and AccessQuick™ RT-PCR Systems using total from genomic DNA will be much larger than the product
RNA template levels in the range of 10pg–1μg per reaction amplified from the target cDNA. This size difference not
(Figure 1.7) or poly(A)+ RNA template levels in the range only makes it possible to differentiate the two products by
of 1pg–100ng. gel electrophoresis but also favors the synthesis of the
smaller cDNA-derived product (PCR favors the
amplification of smaller fragments).
Regardless of primer choice, the final concentration of the
primer in the reaction is usually within the range of
0.1–1.0μM, but this may need to be optimized. We
recommend using a final concentration of 1μM primer
(50pmol in a 50μl reaction) as a starting point for
optimization. More information on PCR primer design is
provided in the PCR Primer Design section.

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D. Cycle Parameters the proofreading function, such as Taq DNA polymerase.
Efficient first-strand cDNA synthesis can be accomplished These two groups have some important differences.
in a 20- to 60-minute incubation at 37–45°C using AMV Proofreading DNA polymerases are more accurate than
reverse transcriptase. We recommend using a nonproofreading polymerases due to the 3′→5′ exonuclease
sequence-specific primer and performing the reverse activity, which can remove a misincorporated nucleotide
transcription reaction at 45°C for 45 minutes as a starting from a growing chain of DNA. When the amplified product
point. The higher temperature will minimize the effects of is to be cloned, expressed or used in mutation analysis, Pfu
RNA secondary structure and encourage full-length cDNA DNA polymerase is a much better choice due to its high
synthesis. First-strand cDNA synthesis with random fidelity. However, for routine PCR, where simple detection
hexamers and oligo(dT) primer should be conducted at of an amplification product is the goal, Taq DNA
room temperature (20–25°C) and 37°C, respectively. polymerase is the most commonly used enzyme because
The Access and AccessQuick™ RT-PCR Systems do not yields tend to be higher with a nonproofreading DNA
require an RNA denaturation step prior to initiation of the polymerase.
reverse transcription reaction. If desired, however, a Amplification with nonproofreading DNA polymerases
denaturation step may be incorporated by incubating a results in the template-independent addition of a single
separate tube containing the primers and RNA template nucleotide to the 3′-end of the PCR product, whereas the
at 94°C for 2 minutes. Do not incubate AMV reverse use of proofreading DNA polymerases results in
transcriptase at 94°C; it will be inactivated. The blunt-ended PCR products (Clark, 1988; Hu, 1993). The
template/primer mixture can then be cooled to 45°C and single-nucleotide overhang can simplify the cloning of PCR
added to the RT-PCR reaction mix for the standard reverse products.
transcription incubation at 45°C. Following the reverse Proofreading DNA polymerases are also used in blends
transcription, we recommend a 2-minute incubation at 94°C with nonproofreading DNA polymerases, or
to denature the RNA/cDNA hybrid, inactivate AMV reverse amino-terminally truncated versions of Taq DNA
transcriptase and dissociate AMV RT from the cDNA. It polymerase, to amplify longer stretches of DNA with
has been reported that AMV reverse transcriptase must be greater accuracy than the nonproofreading DNA
inactivated to obtain high yields of amplification product polymerase alone (Barnes, 1994; Cline et al. 1996). See Long
using thermophilic DNA polymerases such as Tfl DNA PCR.
polymerase (Sellner et al. 1992; Chumakov, 1994).
A. Taq DNA Polymerase
Most RNA samples can be detected using 30–40 cycles of
amplification. If the target RNA is rare or if only a small Taq DNA polymerase is isolated from Thermus aquaticus
amount of starting material is available, it may be necessary and catalyzes the primer-dependent incorporation of
to increase the number of cycles to 45 or 50 or dilute the nucleotides into duplex DNA in the 5′→3′ direction in the
products of the first reaction and reamplify. presence of Mg2+. The enzyme does not possess 3′→5′
exonuclease activity but has a 5′→3′ exonuclease activity.
IV. Thermostable DNA Polymerases
Prior to the use of thermostable DNA polymerases in PCR, Taq DNA polymerase is suitable for most PCR
researchers had to laboriously replenish the reaction with amplifications that do not require a high-fidelity enzyme,
fresh enzyme (such as Klenow or T4 DNA polymerase) such as the detection of specific DNA or RNA sequences.
after each denaturation cycle. Thermostable DNA The error rate of Taq DNA polymerase is approximately 1
polymerases revolutionized and popularized PCR because × 10–5 errors/base, although the fidelity does depend
of their ability to withstand the high denaturation somewhat on the reaction conditions. The fidelity is slightly
temperatures. The use of thermostable DNA polymerases higher at lower pH, lower magnesium concentration and
also allowed higher annealing temperatures, which relatively low dNTP concentration (Eckert and Kunkel,
improved the stringency of primer annealing. 1990; Eckert and Kunkel, 1991). See High-Fidelity PCR.
Thermostable DNA polymerases can also be used for either Taq DNA polymerase is commonly used to amplify PCR
one-enzyme or two-enzyme RT-PCR (Myers and Gelfand, products of 5kb or less. PCR products in the range of 5–10kb
1991; Chiocchia and Smith, 1997). For example, Tth DNA can be amplified with Taq DNA polymerase but often
polymerase can act as a reverse transcriptase in the presence require more optimization than smaller PCR products. For
of Mn2+ for one-enzyme RT-PCR (Myers and Gelfand, 1991). products larger than approximately 10kb, we recommend
All of the DNA polymerases mentioned below can be used an enzyme or enzyme mix and reaction conditions that are
for amplification of the first-strand cDNA produced by a designed for long PCR.
reverse transcriptase, such as AMV RT, in two-enzyme Taq DNA polymerase is a processive enzyme with an
RT-PCR. extension rate of >60 nucleotides/second at 70°C (Innis et
The thermostable DNA polymerases can be divided into al. 1988), so an extension step of 1 minute per 1kb to be
amplified should be sufficient to generate full-length PCR
two groups: those with a 3′→5′ exonuclease (proofreading)
products. The enzyme has a half-life of 40 minutes at 95°C
activity, such as Pfu DNA polymerase, and those without
(Lawyer et al. 1993). Because Taq DNA polymerase is a

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nonproofreading polymerase, PCR products generated advantage over more commonly used reverse
with Taq DNA polymerase will contain a single-nucleotide transcriptases, such as AMV and M-MLV reverse
3′ overhang, usually a 3′ A overhang. transcriptases. Recombinant Tth DNA polymerase has been
shown to exhibit RNase H-like activity (Auer et al. 1995).
Additional Resources for Taq DNA Polymerases
Technical Bulletins and Manuals Additional Resources for Tth DNA Polymerases
9PIM300 GoTaq® DNA Polymerase Promega Product Technical Bulletins and Manuals
Information 9PIM210 Tth DNA Polymerase Promega Product
(www.promega.com Information
/tbs/9pim300/9pim300.html) (www.promega.com
9PIM829 GoTaq® Flexi® DNA Polymerase Promega /tbs/9pim210/9pim210.html)
Product Information D. Tli DNA Polymerase
(www.promega.com
/tbs/9pim829/9pim829.html) Tli DNA polymerase replicates DNA through the
Promega Publications polymerization of nucleotides into duplex DNA in the 5′→3′
enotes GoTaq® Green Master Mix for Quick and direction in the presence of Mg2+. This enzyme also contains
Easy Two-Step RT-PCR a 3′→5′ exonuclease activity, which results in increased
(www.promega.com fidelity of nucleotide incorporation. There is no detectable
/enotes/applications/ap0069.htm) reverse transcriptase activity or 5′→3′ exonuclease activity.
Online Tools Tli DNA polymerase will promote strand displacement at
GoTaq® DNA Polymerase FAQ (www.promega.com 72°C but will not displace DNA at 55°C (Kong et al. 1993).
/faq/gotaq.html) Greater than 95% of the amplified products will be
blunt-ended.
B. Tfl DNA Polymerase
Tli DNA polymerase is commonly used for PCR and
Tfl DNA polymerase catalyzes the primer-dependent
RT-PCR, where its proofreading activity makes it useful
polymerization of nucleotides into duplex DNA in the
for high-fidelity and long PCR (Keohavong et al. 1993). Due
presence of Mg2+. In the presence of Mn2+, Tfl DNA
to the 3′→5′ exonuclease activity of Tli DNA polymerase,
polymerase catalyzes the polymerization of nucleotides
the enzyme can degrade the oligonucleotide primers used
into DNA, using RNA as a template. Tfl DNA polymerase
to initiate DNA synthesis. This exonucleolytic attack can
exhibits a 5′→3′ exonuclease activity but lacks a 3′→5′ be effectively prevented by using hot-start PCR or
exonuclease activity. This enzyme is commonly used in introducing a single phosphorothioate bond at the 3′ termini
PCR (Gaensslen et al. 1992), where its activity is similar to of the primer (Byrappa et al. 1995). Tli DNA polymerase
that of Taq DNA polymerase. Tfl DNA polymerase is used can also be used for primer extension, where the high
in the Access and AccessQuick™ RT-PCR Systems. optimal temperature of the enzyme may be an advantage
C. Tth DNA Polymerase for templates with complex secondary structure.
Tth DNA polymerase catalyzes the polymerization of E. Pfu DNA Polymerase
nucleotides into duplex DNA in the 5′→3′ direction in the Pfu DNA polymerase has one of the lowest error rates of
presence of MgCl2. The enzyme is also capable of catalyzing all known thermophilic DNA polymerases used for
the polymerization of DNA using an RNA template in the amplification due to the highly active 3′→5′ exonuclease
presence of MnCl2 (Myers and Gelfand, 1991; Ruttimann activity (Cline et al. 1996; Andre et al. 1997). For cloning and
et al. 1985). Tth DNA polymerase exhibits a 5′→3′ expressing DNA after PCR, Pfu DNA polymerase is the
enzyme of choice. Pfu DNA polymerase can be used alone
exonuclease activity but lacks detectable 3′→5′ exonuclease
for the amplification of DNA fragments up to 5kb by
activity. The error rate of Tth DNA polymerase has been
increasing the extension time to 2 minutes per kilobase. It
measured at 7.7 × 10–5 errors/base (Arakawa et al. 1996).
is also used in blends with DNA polymerases lacking the
Tth DNA polymerase can amplify target DNA in the
proofreading function, such as Taq DNA polymerase, to
presence of phenol-saturated buffer (Katcher and Schwartz,
achieve longer amplification products than with Pfu DNA
1994) and has been reported to be more resistant to
polymerase alone (Barnes, 1994). However, the
inhibition by blood components than other thermostable
proofreading activity can shorten PCR primers, leading to
polymerases (Ehrlich et al. 1991; Bej and Mahbubani, 1992).
decreased yield and increased nonspecific amplification.
Tth DNA polymerase is commonly used for PCR (Myers This exonucleolytic attack can be effectively prevented by
and Gelfand, 1991; Carballeira et al. 1990) and RT-PCR initiating the reaction using hot-start PCR or by introducing
(Myers and Gelfand, 1991; Chiocchia and Smith, 1997). For a single phosphorothioate bond at the 3′-termini of the
primer extension, RT-PCR and cDNA synthesis using RNA primers (Byrappa et al. 1995).
templates with complex secondary structure, the high
reaction temperature of Tth DNA polymerase may be an

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(42°C) makes it the enzyme of choice for cDNA synthesis
Additional Resources for Pfu DNA Polymerases
using templates with complex secondary structure, its
Technical Bulletins and Manuals relatively high RNase H activity limits its usefulness for
9PIM774 Pfu DNA Polymerase Promega Product generation of long cDNAs (>5kb). For these templates,
Information M-MLV RT, RNase H minus, may be a better choice.
(www.promega.com
/tbs/9pim774/9pim774.html) Additional Resources for AMV Reverse Transcriptase
Promega Publications Technical Bulletins and Manuals
PN068 Pfu DNA Polymerase: A high fidelity 9PIM510 AMV Reverse Transcriptase Promega Product
enzyme for nucleic acid amplification Information
(www.promega.com (www.promega.com
/pnotes/68/7381_07/7381_07.html) /tbs/9pim510/9pim510.html)

V. Reverse Transcriptases B. M-MLV Reverse Transcriptase

The discovery of reverse transcriptases, or RNA-dependent M-MLV RT is a single-polypeptide, RNA-dependent DNA
DNA polymerases, and their role in retrovirus infection polymerase. The enzyme also has DNA-dependent DNA
(Baltimore, 1970; Temin and Mizutani, 1970) altered polymerase activity at high enzyme levels (Roth et al. 1985).
molecular biology’s central dogma of M-MLV RT is used in a variety of applications, including
cDNA synthesis, RT-PCR and RACE (Gerard, 1983). Its
DNA→RNA→protein. Reverse transcriptases use an RNA
relatively low RNase H activity compared to AMV RT
template to synthesize DNA and require a primer for
makes M-MLV RT the enzyme of choice for generating long
synthesis, like other DNA polymerases. For in vitro
cDNAs (>5kb) (Sambrook and Russell, 2001). However, for
applications, the primer can be either oligo(dT), which
short templates with complex secondary structure, AMV
hybridizes to the poly(A)+ tails of eukaryotic mRNAs,
RT or M-MLV RT, RNase H minus, may be better choices
random hexamers, which prime the synthesis from internal
due to their higher optimal temperatures. M-MLV RT is
sites of the RNA template, or a sequence-specific primer,
less processive than AMV RT, so more units of M-MLV RT
which hybridizes to a known sequence within the RNA
may be required to generate the same amount of cDNA
template. Polymerization from a primer then proceeds as
(Schaefer, 1995).
for DNA-dependent DNA polymerases. The commonly
used reverse transcriptases, avian myeloblastosis virus Additional Resources for M-MLV Reverse Transcriptase
reverse transcriptase (AMV RT), Moloney murine leukemia
virus reverse transcriptase (M-MLV RT) and M-MLV Technical Bulletins and Manuals
reverse transcriptase, RNase H minus, perform the same 9PIM170 M-MLV Reverse Transcriptase Promega
reaction but at different optimum temperatures (AMV, Product Information
42°C; M-MLV, 37°C; and M-MLV RT, RNase H–, 42°C). (www.promega.com
/tbs/9pim170/9pim170.html)
Some reverse transcriptases also possess intrinsic 3′- or
5′-exoribonuclease (RNase) activity, which is generally used C. M-MLV Reverse Transcriptase, RNase H Minus
to degrade the RNA template after the first strand of a
M-MLV reverse transcriptase, RNase H minus, is an
cDNA is produced. Absence of the 5′-exoribonuclease
(RNase H) activity may aid in the production of longer RNA-dependent, 5′→3′ DNA polymerase that has been
cDNAs (Berger et al. 1983). genetically altered to remove the associated ribonuclease
H activity, which causes degradation of the RNA strand of
Some DNA-dependent DNA polymerases also possess a an RNA:DNA hybrid (Tanese and Goff, 1988). The absence
reverse transcriptase activity, which can be favored under of RNase H activity makes M-MLV, RNase H minus, the
certain conditions. For example, the thermostable, enzyme of choice for generating long cDNAs (>5kb).
DNA-dependent Tth DNA polymerase exhibits reverse However, for shorter templates with complex secondary
transcriptase activity when Mn2+ is substituted for Mg2+ structure, AMV reverse transcriptase may be a better choice
in a reaction. because it can be used at higher reaction temperatures.
A. AMV Reverse Transcriptase There are two forms of M-MLV, RNase H minus: the
AMV RT catalyzes the polymerization of DNA using deletion mutant and the point mutant. As the names
template DNA, RNA or RNA:DNA hybrids (Houts et al. suggest, the deletion mutant had a specific sequence in the
1979). AMV reverse transcriptase is the preferred reverse RNase H domain deleted, and the point mutant has a point
transcriptase for templates with high secondary structure mutation introduced in the RNase H domain. While the
due to its higher reaction temperature (up to 58°C). AMV native M-MLV RT has a recommended reaction
RT is used in a wide variety of applications including cDNA temperature of 37°C, the deletion and point mutants are
synthesis (Houts et al. 1979; Gubler and Hoffman, 1983), more stable at higher temperatures and can be used at
RT-PCR and rapid amplification of cDNA ends (RACE; reaction temperatures of up to 50°C and 55°C, respectively,
Skinner et al. 1994). Although the high optimal temperature depending upon the reverse transcription primers used.

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The point mutant is often preferred over the deletion • upstream primer
mutant because the point mutant has DNA polymerase • GoTaq® DNA Polymerase (Cat.# M8291)
activity comparable to that of the wildtype M-MLV enzyme, • MgCl2, 25mM
whereas the deletion mutant has a slightly reduced DNA • Nuclease-Free Water (Cat.# P1193)
polymerase activity compared to that of the wildtype • nuclease-free light mineral oil (e.g., Sigma Cat.# M5904)
enzyme (Figure 1.8). if using a thermal cycler without a heated lid; do not
M-MLV RT autoclave
• dNTP mix, 10mM of each dNTP
Point Mutant
Note: To facilitate optimization, troubleshooting and
Deletion Mutant
validation of any PCR reaction, we strongly recommend
RNase H+ including both positive and negative control reactions.
1,000 1. Combine the first five reaction components in the order
cDNA Synthesized (ng)

listed below in a thin-walled 0.5ml reaction tube. Gently

800 vortex the tube for 10 seconds and briefly centrifuge in
a microcentrifuge. Initiate the reaction by adding the
600
template and primers.
400
Final
2919MA03_0A

200 Component Volume Concentration

Nuclease-Free Water (to Xμl
0 a final volume of 50μl)
0 200 400
5X Green or Colorless 10μl 1X
Units of Enzyme GoTaq® Flexi Buffer
Figure 1.8. Comparison of the mass amount of total cDNA dNTP mix, 10mM each 1μl 0.2mM each
synthesized from 2μg of a 7.5kb RNA template by increasing dNTP
amounts of three Promega M-MLV reverse transcriptases. Each GoTaq® DNA 0.25μl 0.025u/μl
first-strand cDNA reaction was performed using 2μg of a 7.5kb Polymerase (5u/μl)
RNA template (1μl), 0.5μg of oligo(dT)15 primer (1μl) and 14μl
25mM MgCl2 3μl 1.5mM
water. The RNA and oligo(dT)15 primer were heated at 70°C for
5 minutes and cooled on ice for 5 minutes. Five microliters of downstream primer 50pmol1 1μM
M-MLV RT 5X Buffer, 1.25μl of 10μM dNTPs, 0.5μl of α-32P dCTP upstream primer 50pmol 1μM
(10μCi/μl, 400Ci/mmol) and either 25, 50, 100, 150, 200 or 400 units Yμl
template2
of M-MLV Reverse Transcriptase, RNase H Minus, Point Mutant;
M-MLV Reverse Transcriptase, RNase H Minus, Deletion Mutant; 1
A general formula for calculating the number of nanograms of
or native M-MLV Reverse Transcriptase (RNase H+) in a final
volume of 25μl. Reactions were incubated at 42°C for 60 minutes. primer equivalent to 50pmol is: 50pmol = 16.3ng × b; where b is
TCA precipitations were performed, and first-strand cDNA yields the number of bases in the primer.
were calculated. 2
If possible, start with >104 copies of the target sequence to obtain
a signal in 25–30 cycles, but keep the final DNA concentration of
Additional Resources for M-MLV Reverse Transcriptase, the reaction at ≤10ng/μl. Less than 10 copies of a target can be
RNase H Minus amplified (Saiki, 1988), but more cycles may be required to detect
Technical Bulletins and Manuals a signal by gel electrophoresis. Additional cycles may increase
9PIM530 M-MLV Reverse Transcriptase, RNase H nonspecific amplification, evidenced by smeared bands upon gel
Minus, Promega Product Information electrophoresis.
(www.promega.com 2. Overlay the reaction with 1–2 drops (20–40μl) of
/tbs/9pim530/9pim530.html) nuclease-free mineral oil to prevent condensation and
9PIM368 M-MLV Reverse Transcriptase, RNase H evaporation. Mineral oil addition is not necessary if
Minus, Point Mutant, Promega Product you are using a thermal cycler with a heated lid.
Information
(www.promega.com 3. Place the tubes in a thermal cycler and proceed with
/tbs/9pim368/9pim368.html) the thermal cycling profile chosen for your reactions.

4. Analyze 5μl of the PCR products by agarose gel

VI. Example of a PCR Protocol electrophoresis. The products should be readily visible
Materials Required: by UV transillumination of the ethidium
(see Composition of Solutions section) bromide-stained gel.
• template DNA
• downstream primer 5. Store reaction products at –20°C until needed.

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VII. Example of an RT-PCR Protocol 3. Place the tubes in a thermal cycler equilibrated at 45°C,
and incubate for 45 minutes.
A. Access RT-PCR Protocol
These conditions work well for the detection of the 323bp 4. Proceed directly to thermal cycling the reactions for
PCR product generated from the Positive Control RNA second-strand cDNA synthesis and amplification (refer
using the Upstream and Downstream Control Primers to Tables 1.1 and 1.2).
provided with the Access RT-PCR System. We recommend
optimizing the parameters for each target RNA. Table 1.1. First-Strand cDNA Synthesis.
1 cycle 45°C for 45 reverse transcription
Materials Required: minutes
(see Composition of Solutions section)
• template RNA 1 cycle 94°C for 2 AMV RT inactivation
• downstream oligonucleotide primer minutes and
RNA/cDNA/primer
• upstream oligonucleotide primer denaturation
• Access RT-PCR System (Cat.# A1250)
• Nuclease-Free Water (Cat.# P1193) Table 1.2. Second-Strand cDNA Synthesis and PCR.
• nuclease-free light mineral oil (e.g., Sigma Cat.# M5904) 40 cycles 94°C for 30 seconds denaturation
if using a thermal cycler without a heated lid 60°C for 1 minute annealing
1. Prepare the reaction mix by combining the indicated 68°C for 2 minutes extension
volumes of Nuclease-Free Water, AMV/Tfl 5X Reaction 1 cycle 68°C for 7 minutes final
Buffer, dNTP Mix, 25mM MgSO4 and the specific extension
upstream and downstream primers in a thin-walled 1 cycle 4°C soak
0.5ml reaction tube on ice. Mix the components by
B. ImProm-II™ Reverse Transcription System Protocol
pipetting. Add the AMV Reverse Transcriptase and Tfl
DNA Polymerase to the reaction. Gently vortex the tube 1. Place sterile, thin-walled dilution tubes and reaction
for 10 seconds to mix the components. tubes on ice. Thaw the experimental RNA or the 1.2kb
Kanamycin Positive Control RNA on ice and return
Final any unused portion to the freezer as soon as aliquots
Component Volume Concentration are taken.
Nuclease-Free Water (to Xμl
a final volume of 50μl) 2. On ice, combine the RNA (up to 1μg) and the cDNA
AMV/Tfl 5X Reaction 10μl 1X primer in Nuclease-Free Water for a final volume of
Buffer 5μl per reaction.
dNTP Mix, 10mM each 1μl 0.2mM each
dNTP Experimental Reactions
downstream primer 1μM Component Volume
50pmol3
Experimental RNA (up to 1μg/reaction)5 Yμl
upstream primer 50pmol 1μM
Oligo(dT)15 Primer or Random Primers Xμl
25mM MgSO4 2μl 1mM
(0.5μg/reaction) or gene-specific primer
AMV Reverse 1μl 0.1u/μl (10–20pmol/reaction)6
Transcriptase (5u/μl)
Nuclease-Free Water to a final volume of 5μl
Tfl DNA Polymerase 1μl 0.1u/μl
(5u/μl) 5
102–1010 copies of a specific target RNA template or 1pg–1μg
RNA sample4 Yμl total RNA or poly(A)+ mRNA.
3 6
A general formula for calculating the number of nanograms of 10–20pmol of primer in a 20μl reaction is equal to 0.5–1μM. A
primer equivalent to 50pmol is: 50pmol = 16.3ng × b; where b is general formula for calculating nanograms of primer equivalent
the number of bases in the primer. For the positive control reaction, to 10pmol is 3.3 × b, where b is the number of bases in the primer.
use 3.3μl of both the Downstream and Upstream Control Primers Positive Control Reaction
(50pmol). Component Volume
4 1.2kb Kanamycin Positive Control RNA, 2μl
This is equivalent to 103–106 copies of a specific target template
0.5μg/μl
or 1pg–1μg total RNA. Use 2μl of the Positive Control RNA with
Carrier (2.5 attomoles or 1 × 106 copies). Oligo(dT)15 Primer, 0.5μg/μl 1μl
2. Overlay the reaction with one or two drops (20–40μl) Nuclease-Free Water 2μl
of nuclease-free mineral oil to prevent condensation Final Volume 5μl
and evaporation. Mineral oil addition is not necessary
if you are using a thermal cycler with a heated lid.

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Negative (No Template) Control Reaction Negative (No Reverse Transcriptase) Control Reaction
Component Volume Component Amount
Oligo(dT)15 Primer or Random Primers Xμl Nuclease-Free Water (to a final volume Xμl
(0.5μg/reaction) or gene-specific primer of 15μl)
(10–20pmol/reaction) ImProm-II™ 5X Reaction Buffer 4.0μl
Nuclease-Free Water to a final volume of 5μl MgCl2, 25mM (1.5–8.0mM final conc.) 1.2–6.4μl
3. Close each tube of RNA tightly. Place the tubes into a dNTP Mix, 10mM each dNTP (0.5mM 1.0μl
preheated 70°C heat block for 5 minutes. Immediately final conc.)
chill in ice-water for at least 5 minutes. Spin each tube RNasin® Ribonuclease Inhibitor (optional) 20u
for 10 seconds in a microcentrifuge to collect the Final Volume 15.0μl
condensate and maintain the original volume. Keep the
tubes closed and on ice until the reverse transcription 5. Dispense 15μl of the Reverse Transcription Reaction
reaction mix is added. Mix to each reaction tube on ice. Be careful to prevent
cross-contamination. Add 5μl of RNA and primer mix
4. Prepare the reverse transcription reaction mix by to each reaction, giving a final reaction volume of 20μl
combining the following components of the per tube. If there is a concern about evaporation in
ImProm-II™ Reverse Transcription System in the order subsequent steps, overlay the reaction with a drop of
listed in a sterile 1.5ml microcentrifuge tube on ice. nuclease-free mineral oil to prevent evaporation and
Determine the volume of each component needed for condensation.
the desired number of reaction and combine the
components in the order listed. Vortex gently to mix, 6. Anneal: Place the tubes in a controlled-temperature
and keep on ice prior to dispensing into the reaction heat block equilibrated at 25°C and incubate for 5
tubes. minutes.

Experimental Reactions 7. Extend: Incubate the tubes in a controlled-temperature

Component Volume heat block at 42°C for up to one hour. The extension
Nuclease-Free Water (to a final volume Xμl temperature may be optimized between 37–55°C.
of 15μl)
8. Inactivate reverse transcriptase: If the experimental
ImProm-II™ 5X Reaction Buffer 4.0μl goal is to proceed with PCR, the reverse transcriptase
MgCl2, 25mM (1.5–8.0mM final conc.) 7 1.2–6.4μl must be thermally inactivated prior to amplification.
dNTP Mix, 10mM each dNTP (0.5mM 1.0μl Incubate the reaction tubes in a controlled-temperature
final conc.)8 heat block at 70°C for 15 minutes.
RNasin® Ribonuclease Inhibitor (optional) 20u 9. Prepare the PCR mix by dispensing the appropriate
ImProm-II™ Reverse Transcriptase 1.0μl volume of each component into a sterile, 1.5ml
Final Volume 15.0μl microcentrifuge tube on ice. Combine the components
in the order listed, vortex gently to mix, and keep on
7 ice prior to dispensing to the reaction tubes. An aliquot
The final Mg2+ concentration should be optimized in the range
of 1.5–8.0mM. of the first-strand cDNA (1μl or 20μl) from the reverse
8 transcription reaction is added last to the PCR Mix to
If isotopic or nonisotopic incorporation is desired to monitor this
give a final reaction volume of 100μl per tube. Overlay
first-strand cDNA synthesis, α[32P]-dCTP or other modified
nucleotides may be supplemented into the dNTP mixture.
Positive Control Reaction
Component Volume
Nuclease-Free Water (to a final volume Xμl
of 15μl)
ImProm-II™ 5X Buffer 4.0μl
MgCl2, 25mM (6mM final conc.) 4.8μl
dNTP Mix, 10mM each dNTP (0.5mM 1.0μl
final conc.)
RNasin® Ribonuclease Inhibitor (optional) 20u
ImProm-II™ Reverse Transcriptase 1.0μl
Final Volume 15.0μl

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the reaction with two drops of nuclease-free mineral Table 1.3. Amplification Conditions for the Positive
oil to prevent evaporation and condensation. See Notes Control Reaction.
1–3. Denaturation: 94°C for 2 minutes
25 cycles:
Volume Volume
per 100μl per 100μl Denaturation: 94°C for 1 minute
reaction reaction Annealing: 60°C for 1 minute
(1μl RT (20μl RT
Component reaction) reaction) Extension: 72°C for 2 minutes
Nuclease-Free Water 55.2μl 45.6μl Final extension: 72°C for 5 minutes
5X Green or Colorless 19.8μl 16.0μl Hold 4°C
GoTaq® Flexi Buffer
MgCl2, 25mM (2mM final 7.8μl 3.2μl 11. After the cycle is complete, analyze the products or
store the amplifications at –20°C.
conc.)9
PCR Nucleotide Mix, 2.0μl 1.0μl 12. Analyze the PCR reaction products by agarose gel
10mM (0.2mM final conc.) electrophoresis of 10% of the total reaction. The
Upstream Control Primer 6.6μl 6.6μl products will be readily visible by UV transillumination
(1μM final conc.) of an ethidium bromide-stained gel. The amplification
Downstream Control 6.6μl 6.6μl product obtained using the Positive Control RNA with
Primer (1μM final conc.) the Upstream and Downstream Control Primers is
GoTaq® DNA Polymerase 1.0μl 1.0μl 323bp long.
(5.0 units)
PCR Mix per reaction 99μl 80μl 13. Store the reaction products at –20°C until needed.

RT reaction per reaction 1.0μl 20.0μl Notes

Total PCR Volume 100.0μl 100.0μl 1. In this example, the final volume of PCR Mix should
be sufficient for 100μl reactions once the cDNA volume
9 is added. The volume of each component may be scaled
For experimental systems, the final Mg2+ concentration should
be optimized in the range of 1.5–2.5mM. for reactions of less than 100μl. Scale up the volumes
10. Place the reactions in a thermal cycler that has been to accommodate the total number of PCR amplifications
prewarmed to 94°C. An optimized program for being performed.
amplification using the Upstream and Downstream 2. The amount of reverse transcription reaction used in
Control Primers provided with this system is given in the PCR may be modified after experimental
Table 1.3. optimization.
3. Because of the ionic conditions, magnesium
concentration and dNTP concentration of the reverse
transcription reaction, the amount of magnesium and
dNTP added to the PCR varies, depending on how
much RT reaction is used as template. For example, for
a 100μl PCR that contains 20μl of RT product, 8μl of
10X thermophilic polymerase reaction buffer is added
to support the 80μl PCR Mix addition. If 5μl of RT
reaction were added to 95μl of PCR Mix, 9.5μl of 10X
thermophilic polymerase reaction buffer would be
needed. Similar considerations must be given to the
magnesium and dNTP additions.

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VIII.Troubleshooting PCR and RT-PCR
Symptoms Solutions
Low yield or no amplification product Template is degraded. Verify the integrity of the template by
(PCR or RT-PCR) electrophoresis after incubation. Repurify the DNA or RNA template
if the nucleic acid appears degraded.
Inhibitor is present in sample. Reduce the volume of template in the
reaction. Ethanol precipitate to remove inhibitors. Some of the common
inhibitors are listed in the Template Quality section.
Poor primer design. Make sure primers are not self-complementary
or complementary to each other.
Verify that the primers are complementary to the appropriate strands.
Insufficient number of cycles. Return reactions to thermal cycler for 5
more cycles.
Primer concentration is too low. Verify primer concentration in the
reaction. Increase primer concentration if necessary.
Suboptimal reaction conditions. Optimize Mg2+ concentration,
annealing temperature and extension time. Verify that primers are
present in equal concentration. Refer to General Considerations for
PCR Optimization for more information about optimizing reaction
conditions.
Nucleotides are degraded. Keep nucleotides frozen in aliquots, thaw
quickly and keep on ice once thawed. Avoid multiple freeze-thaw
cycles.
Target sequence is not present in target DNA. Redesign experiment
or try other sources of target DNA.
Reaction component is missing. Always perform a positive control
reaction with a template/primer combination that has amplified well
in the past to determine when a component was omitted. Check the
reaction components and repeat the reaction.
Poor-quality mineral oil. The reaction must be overlaid with
high-quality, nuclease-free light mineral oil when using a thermal
cycler without a heated lid. Do not use autoclaved mineral oil.
Thermal cycler was programmed incorrectly. Verify that times and
temperatures are correct. Use step cycles, not hold segments.
Thermal cycler is not reaching the proper temperature. Calibrate the
thermal cycler to be sure the reactions are heated to the programmed
temperatures. Depending upon the primers and template, small
changes in cycling conditions can affect yield.
Temperature is too low in some positions of thermal cycler. Perform
a set of control reactions to determine if certain positions in the thermal
cycler give low yields.
Nonspecific amplification products (PCR Reaction conditions are suboptimal. Optimize Mg2+ concentration,
or RT-PCR) annealing temperature, size, extension time and cycle number to
minimize nonspecific priming. Refer to General Considerations for
PCR Optimization for more information about optimizing reaction
conditions.
Poor primer design. Make sure primers are not self-complementary
or complementary to each other, especially near the 3′-ends. Avoid
using three G or C nucleotides in a row at the 3′-end of a primer. Try
a longer primer.
Primer concentration is too high. Verify primer concentration in the
reaction. Try a lower concentration in the reaction.
Reaction is contaminated by another RNA/DNA. Use positive
displacement pipets or aerosol-resistant tips to reduce
cross-contamination during pipetting. Use a separate work area and
pipettor for pre- and postamplification. Wear gloves and change them
often. Use UNG or another technique to prevent carryover of DNA
produced in a previous amplification into subsequent reactions. See
the Nucleic Acid Cross-Contamination section.
Multiple target sequences exist. Design new primers with higher
specificity to target sequence in template DNA or cDNA.

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Symptoms Solutions
Low yield or no first-strand product RNA is degraded. Verify the RNA integrity by denaturing agarose gel
(RT-PCR) electrophoresis. Ensure that reagents, tips and tubes are RNase-free.
Isolate the RNA in the presence of a ribonuclease inhibitor (e.g.,
Promega RNasin® Ribonuclease Inhibitor). Repurify the DNA or RNA
template if the nucleic acid appears degraded.
AMV Reverse Transcriptase was thermally inactivated. If an initial
denaturation/annealing step is introduced into the protocol, be certain
to add the enzyme mix containing AMV Reverse Transcriptase after
the denaturation step and subsequent 45°C equilibration.
Poor primer specificity. Verify that the reverse transcription primer is
complementary to the downstream sequence of the RNA.
Poor primer annealing. If oligo(dT) primers or random hexamers were
used as the reverse transcription primer, verify that the annealing step
was carried out at an appropriate temperature prior to reverse
transcription.
RNA template is impure. Carryover of reagents (e.g., SDS, NaCl,
heparin, guanidine thiocyanate) from some RNA purification methods
can interfere with RT-PCR. Reduce volume of target RNA, perform
additional purification steps or change purification method.
Amplification product with a Genomic DNA sequences related to the RNA template contaminate
higher-than-expected molecular weight the RNA preparation. Treat the RNA sample with RQ1 RNase-Free
(RT-PCR) DNase to degrade contaminating DNA.

yield of single-stranded complementary deoxyribonucleic acid.

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| 1Nucleic Acid Amplification

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temperature triggering for the polymerase chain reaction. Products may be covered by pending or issued patents or may have certain
limitations. Please visit our Web site for more information.
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All prices and specifications are subject to change without prior notice.
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genetic alphabet to quench fluorescence. J. Am. Chem. Soc. 126, Services or access the Promega online catalog for the most up-to-date
information on Promega products.
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© 2004–2006 Promega Corporation. All Rights Reserved.
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