Polymerase Chain Reaction

(PCR): Specialized Reactions

Stella E Tsirka, State University of New York, Stony Brook, New York, USA
Michael A Frohman, State University of New York, Stony Brook, New York, USA

Secondary article
Article Contents
. Introduction
. Detection of Gene Expression
. Sequencing
. Detection of Mutations and Genotyping

The polymerase chain reaction is a technique that rapidly amplifies selected subsets of DNA
or complementary DNA from initially complex biological mixtures. The efficiency of
amplification or the sequence of the amplified material can then be examined for any of
many purposes, including genotyping or the characterization of new genes, gene
expression patterns, mutations or polymorphisms.

Introduction
An individual gene comprises a very small part of any
genome. Methods to identify or to examine the structure of
individual genes from complex cellular mixtures containing tens to hundreds of thousands of genes were developed
in the 1970s, but were laborious, often requiring weeks or
months to complete. Development of the polymerase chain
reaction (PCR) resulted in the ability to selectively amplify
individual genes out of these complex mixtures, following
which the genes could then be rapidly analysed without
further purication. Numerous classical techniques have
since been adapted to incorporate the power of PCR,
permitting such investigations to be performed in often a
tenth to a one-hundredth of the original time.

RACE
RACE rapid amplication of complementary DNA
(cDNA) ends constitutes a method through which
individual cDNAs for which only part of the sequence is

known can be amplied and analysed from complex

mixtures of cellular mRNA (Frohman et al., 1988). Most
genes that are expressed in cells are found at relatively low
levels of abundance, on the order of 110 copies per cell.
Since the average cell contains hundreds of thousands of
mRNAs, this typically meant that investigators had to
screen upwards of one million cDNA clones to ensure a
reasonable chance of nding a given gene. The eort
required to do this was substantial and costly.
In contrast, RACE can be used to amplify a cDNA
containing both known and unknown sequence to the
point where it can be analysed directly without cloning.
The classic scheme for this is shown in Figure 1. PCR
consists of repeated cycles of copying of DNA or cDNA
templates between two oligonucleotide primers of known
sequence that promote synthesis towards each other. Since
in this case part of the sequence is unknown, the key step in
RACE involves incorporating or appending a tag on to
the end of the cDNA for which the sequence is unknown.
Oligonucleotide primers complementary to this tag are
then used in conjunction with primers specic to the gene of

mRNA
AAAA
GSP-RT
Reverse transcription

to copy mRNA into cDNA

AAAA

1st strand cDNA

cDNA tailing to

GSP-RT
append a tag at the unknown end
AAAA

AAAA

1st strand cDNA

GSP-RT

Amplification of cDNA end using T-tailed universal primer

universal-TTTT
GSP1
Figure 1 Schematic representation of classic RACE. GSP1, gene-specific primer 1; GSP-RT, gene-specific primer used for reverse transcription.

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Polymerase Chain Reaction (PCR): Specialized Reactions

mRNA
AAAA
Removal of cap from

5' end of mRNA

mRNA
AAAA
Ligation of universal RNA
oligo to 5' end of mRNA
mRNA
GSP-RT
universal

universal

Reverse transcription
1st strand cDNA

AAAA

to generate cDNA
GSP-RT

Amplification
GSP1

Figure 2 Schematic representation of new RACE. This method differs from classic RACE in that the tag (an RNA oligo) is appended to the mRNA prior to
reverse transcription. Accordingly, only cDNAs that are successfully extended all the way to the 5 end of the mRNA encode the tag and thus become
templates capable of being amplified by the RACE primers.

interest to amplify the intervening fragment. Background

amplication can occur in PCR reactions for many
reasons. Accordingly, an optional part of the RACE
technique is to use nested primers, which contribute a
second level of specicity to the reaction and eliminate all
but the desired amplied fragment from the nal reaction
product.
The major historical limitation of RACE involved the
rst step, conversion of cellular mRNA to cDNA. In many
cases, the reverse transcription became stalled, leading to
the majority of cDNAs being less than fully extended
relative to the end of the original mRNA. Many groups
have since proposed a solution to this, which is to append
the universal tag to the end of the mRNA prior to reverse
transcription (Figure 2); accordingly, only those cDNAs
that become extended all the way to the end of the mRNA
encode the tag, which is required for amplication (Zhang
and Frohman, 1999). Current research on RACE technology involves increasing the eciency of appending this tag
using RNA ligase or tags that fuse to the mRNA cap.
In addition to gene identication, RACE is also useful
for examining usage of alternate promoter initiation and
polyadenylation signal sites.

Detection of Gene Expression

Classical techniques for examining patterns of gene
expression required large numbers of cells as starting
material in order to generate sucient mRNA that
transcripts from individual genes could be detected by
hybridization after electrophoresis in agarose gels. PCR
oers the ability to amplify and detect the mRNA-derived
signal from minute samples (for example, one cell). This is
2

especially useful for low-abundance samples, such as

discrete regions in early embryos or the adult brain, or
small foci of tumour cells microdissected away from
normal tissue.
The technique involves converting the mRNA into
cDNA using reverse transcriptase, and then amplifying the
cDNA using two oligonucleotide primers of known
sequence located a short distance apart that cause the
intervening region to be repeatedly copied. Eventually such
amplication hits a plateau, once a certain amount of
product has been generated (Figure 3). The classic detection
method involved performing the amplication for a limited
number of cycles, such that the reaction was stopped before
the plateau was reached. The products were then separated
by electrophoresis on agarose gels and quantied using
direct (ethidium bromide) or indirect (hybridization)
means. Unfortunately, it is hard in practice to predict
when the plateau will be reached, and this event generally is
dierent for each sample, depending on the initial amount
of template and the eciency of the amplication process.
Moreover, it could not be determined, by looking at a
single point, whether the reaction had been stopped in the
exponential phase or whether it had already reached the
plateau. Finally, the sensitivity of detection of PCR
products using these methods was suciently limited that
the products often could not be observed until their
amplication level had actually begun to reach the plateau.
Current state-of-the-art approaches involve measuring
the amount of amplication product at the end of each
cycle of the reaction, which ensures that estimates of the
concentration are made during the exponential phase of
amplication (Schalasta and Schmid, 1999). This method
is rapid and very precise; using it, small dierences in levels
of gene expression can be demonstrated in less than an
hour. Two methods are popularly used to monitor real-

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Amount of PCR product (log)

Polymerase Chain Reaction (PCR): Specialized Reactions

10

15

20

25

30

Number of cycles
Figure 3 Accumulation of PCR products during successive cycles of
amplification. Amplification profiles are shown for two samples that differ
8-fold in the starting quantity of template. Note that although the 8-fold
difference is easily discerned during the logarithmic phase of amplification
for both samples (i.e. within the dotted box), examining the output at cycle
26 (arrow) or later would lead to the incorrect conclusion that the original
samples had contained equal starting amounts of material, because all
amplifications eventually cease at the same plateau.

time increases in PCR products. One uses the dye SYBR

green I, which uoresces at a specic wavelength only when
bound to double-stranded DNA (not when bound to the
single-stranded nucleotides, primers or denatured template; Morrison et al., 1998). Thus, this dye generates a
signal that is proportional to the amount of PCR product
generated. The other method employs modied primers
(molecular beacons) that have uorescent tags attached
to them that are initially quenched, i.e. do not emit light,
but become able to do so once the primers are incorporated
into the PCR product (Tyagi and Kramer, 1996).

the sequencing reaction is repeated 25 times while cycling

during the course of the experiment. Second, decreasing
the amount of template greatly decreases its opportunity to
reanneal before the primers have had a chance to bind.
Finally, the use of thermostable polymerases allows the
primer annealing and extension steps to be performed at
elevated temperatures that again minimize reannealing of
the template, and in addition greatly decrease the negative
eect of (G+C)-rich secondary structures in the DNA
template that interfere with sequencing at lower temperatures. In modern techniques, PCR sequencing is generally
conducted using (termination) nucleotides labelled with
uorescent tags, which are compatible with automated
sequencing machines and which can be detected faster and
with greater sensitivity than radioactively tagged nucleotides.
The small amount of template required in PCR
sequencing ultimately makes possible new types of
sequencing applications. For example, very large templates phage, BAC, and YAC vectors up to hundreds of
thousands of base pairs in length can be sequenced
without further subcloning. Moreover, the ability to
sequence small linear fragments (such as PCR products)
underlies techniques under development to assemble
integrated single machines capable of taking a cellular
sample, such as bacteria or human epithelial cells obtained
from rinsing out ones mouth, and processing it to extract
the DNA, amplify a specic gene fragment, and obtain
sequence information. This technology will permit rapid
identication of bacterial strains and human allelic
variations/mutations in the eld or clinical setting. In the
laboratory setting, sequencing of PCR fragments is used
routinely for analysis after amplication of gene fragments
from individuals in which a mutation is suspected.

Detection of Mutations and Genotyping

Sequencing
Classical methods for sequencing generally required the
DNA fragment under investigation to be subcloned and
amplied in a bacterial host in order to generate enough
template for the sequencing reaction. In addition, it was
very challenging to attempt to sequence small linear gene
fragments (such as PCR products) because, at the high
concentrations of template required to generate a detectable signal using radioactively labelled nucleotides and Xray lm, and using the relatively low temperatures (378C)
required for standard Escherichia coli or phage DNA
polymerases, the denatured DNA (single-strand) templates almost completely reannealed to form duplex DNA
before primers could anneal to them and become extended.
PCR sequencing accordingly oers several advantages.
First, a very small amount of template can be used, since

PCR-based methods have been developed to permit

detection of mutations predictive for specic diseases,
identication of alleles in population surveys, and separation of allele-specic DNA strands prior to sequencing. A
PCR-based version of denaturing gradient gel electrophoresis (DGGE) has proved to be the most popular
method, since it is rapid and very sensitive. DGGE takes
advantage of the dierence in the melting temperature of
double-stranded DNA that occurs when a single base pair
is changed. Accordingly, individual alleles of a specic
DNA (PCR) fragment will migrate dierently on DGGE,
since they will melt at slightly dierent points along the
electrophoresis gradient, and double-stranded DNA migrates at a dierent speed from single-stranded or partially
melted DNA. PCR is also used to append long stretches of
G-C repeats (GC-clamps) to the end of the DNA fragments

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Polymerase Chain Reaction (PCR): Specialized Reactions

Wild-type allele

Mutant allele
*
*
wt

mut

wt

mut

PCR products
*Site of mutation that
creates a new restriction site

PCR product

After restriction digest

Figure 4 Schematic representation of PCR-genotyping, when a new restriction site has been introduced in the mutant allele.

(by incorporating them into the primers) to allow the entire

fragment to be assessed for polymorphisms.
In addition to detecting mutations, PCR is being used
extensively to map and detect new genes with the help of
chromosomal sequence-tagged sites (STS). These sites,
generally the ends of large BAC or YAC clones, are short
DNA sequences and correspond to unique regions within
the genome. Specic STS primer pairs and the sequences
they amplify are stored in electronic databases, generating
a common database for genome mapping.
Identication of quantitative trait loci (QTL) relies on
polymorphisms detected between two dierent populations of the same species. These two populations must
show extremely dierent phenotypes/behaviours in response to a specic experimental paradigm. Using PCR
and sets of primers corresponding to dierent chromosomal locations, one can predict that a chromosomal region
is associated with a phenotypic trait, and eventually isolate
the gene(s) responsible for the trait.
Another very ecient and time-saving application based
on PCR is ospring genotyping, which in the past involved
relatively large amounts of DNA, radioactivity, and a few
days of work involving Southern blot analysis. Now,
however, using specic primers and PCR, the detection of a
fragment corresponding to an inserted gene (such as the
neo cassette introduced in knockout mice) is greatly
facilitated. In the case of a single nucleotide change, a
more involved combination of two successive PCR
amplications (using nested primers, the second amplication of which succeeds or fails depending on the genotype),
or a PCR amplication followed by hybridization (at
temperatures that would allow annealing of the wild-type
oligonucleotide, but not the mutant), or restriction digest

(if a new site is introduced or lost; see Figure 4) is required.

There are also PCR-like techniques, such as the ligation
chain reaction, which generate products only when the
primers specically match the genotype, but these
approaches have not proved as popular.

References
Frohman MA, Dush MK and Martin GR (1988) Rapid production of
full-length cDNAs from rare transcripts by amplication using a single
gene-specic oligonucleotide primer. Proceedings of the National
Academy of Sciences of the USA 85: 89989002.
Morrison TB, Weis JJ and Wittwer CT (1998) Quantication of lowcopy transcripts by continuous SYBR Green I monitoring during
amplication. BioTechniques 24: 954962.
Schalasta G and Schmid M (1999) Ultrarapid and semiautomated realtime PCR a breakthrough in nucleic acid analysis. Clinical
Laboratory 45: 661663.
Tyagi S and Kramer FR (1996) Molecular beaconsprobes that uoresce
upon hybridization. Nature Biotechnology 14: 303308.
Zhang Y and Frohman MA (1999) Rapid amplication of cDNA ends
(RACE) to obtain full length cDNAs. In: Walker J (ed.) The Nucleic
Acids Protocols Handbook. Totowa, NJ: Humana Press.

Further Reading
Letts VA, Felix R, Biddlecome GH et al. (1998) The mouse stargazer
gene encodes a neuronal Ca2 1 -channel gamma subunit. Nature
Genetics 19: 340347.
Louis C, Madueno E, Modolell J et al. (1997) One-hundred and ve new
potential Drosophila melanogaster genes revealed through STS
analysis. Gene 195: 187193.
Spelman R and Bovenhuis H (1998) Genetic response from marker
assisted selection in an outbred population for diering marker
bracket sizes and with two identied quantitative trait loci. Genetics
148: 13891396.

ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Macmillan Publishers Ltd, Nature Publishing Group / www.els.net