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Polymerase Chain Reaction (PCR)Paul C Winter, Belfast City Hospital, Belfast, UK

The polymerase chain reaction is a technique that allows DNA molecules of interest

(usually gene sequences) to be copied in a simple enzyme reaction producing a

sufficient quantity of the copied DNA for detailed analysis or manipulation. The

method is a basic tool in molecular biology with widespread applications in

biological and medical research.

Principle of the PCR

The polymerase chain reaction (PCR) is a powerfuland widely used technique that has greatly advancedour ability to analyze genes. Genomic deoxyribo-nucleic acid (DNA) present in cells contains manythousands of genes. This makes it difcult to isolateand analyze any individual gene. PCR allows specicDNA sequences, usually corresponding to genes orparts of genes, to be copied from genomic DNA in asimple enzyme reaction. The only requirement is thatsome of the DNA sequence at either end of the regionto be copied is known. DNA corresponding to thesequence of interest is copied or amplied by PCRmore than one million fold and becomes the predom-inant DNAmolecule in the reaction. Sufcient DNA isobtained for detailed analysis or manipulation of theamplied gene.

Components of the PCR

DNA is amplied by PCR in an enzyme reaction thatundergoes multiple incubations at three differenttemperatures. Each PCR has four key components.

Template DNA

This contains the DNA sequence to be amplied. Thetemplate DNA is usually a complex mixture of manydifferent sequences, as is found in genomic DNA, butany DNA molecule that contains the target sequencecan be used. Ribonucleic acid (RNA) can also be usedfor PCR by rst making a DNA copy using the enzymereverse transcriptase.

Oligonucleotide primers

Each PCR requires a pair of oligonucleotide primers.These are short single-stranded DNA molecules(typically 20 bases) obtained by chemical synthesis.Primer sequences are chosen so that they bind bycomplementary base pairing to opposite DNA strandson either side of the sequence to be amplied.

DNA polymerase

A number of DNA polymerases are used for PCR. Allare thermostable and can withstand the high temper-atures (up to 100C) required. The most commonlyused enzyme is Taq DNA polymerase from Thermusaquaticus, a bacterium present in hot springs. The roleof the DNA polymerase in PCR is to copy DNAmolecules. The enzyme binds to single-stranded DNAand synthesizes a new strand complementary to theoriginal strand. DNA polymerases require a shortregion of double-stranded DNA to get started. InPCR, this is provided by the oligonucleotide primers,which create short double-stranded regions by bindingon either side of the DNA sequence to be amplied. Inthis way the primers direct the DNA polymerase tocopy only the target DNA sequence.

Deoxynucleotide triphosphates

These molecules correspond to the four bases presentin DNA (adenine, guanine, thymine and cytosine) andare substrates for the DNA polymerase. Each PCRrequires four deoxynucleotide triphosphates (dNTPs)(dATP, dGTP, dTTP, dCTP), which are used by theDNA polymerase as building blocks to synthesize newDNA.

How the PCR Works

PCR allows the amplication of target DNAsequences through repeated cycles of DNA synthesis(Figure 1). Each molecule of target DNA synthesizedacts as a template for the synthesis of new targetmolecules in the next cycle. As a result, the amount oftarget DNA increases with each cycle until itbecomes the dominant DNA molecule in the reaction.During the early cycles, DNA synthesis increasesexponentially but in later cycles, as the amount of

Introductory article

Article contents

Principle of the PCR Components of the PCR How the PCR Works Variations of the PCR Method Applications of the PCR

doi: 10.1038/npg.els.0005339

Polymerase Chain Reaction (PCR)

ENCYCLOPEDIA OF LIFE SCIENCES & 2005, John Wiley & Sons, Ltd. www.els.net 1

Cycle 1

Cycle 2

Cycle 3

Target sequence

94C Denaturation

4565C Annealing

72C Extension

94C

72C

94C

4060C

Primer 1Primer 2

Template DNA(double-stranded)

Template DNA(single-stranded)

Primers bindsingle-strandedtemplate DNA

Template DNA

Template DNA

Primers bindall strands

Template DNALong productsTarget sequence

DNA moleculesexactly matchingtarget sequenceare produced

New DNA

TaqTaqNew DNA

Figure 1 Polymerase chain reaction involves repeated cycles of incubation at three temperatures. The reaction is initially heated to

above 90C. At this temperature, the double-stranded DNA template is denatured and becomes single-stranded. The temperature is nextreduced to 4565C. At this temperature, the oligonucleotide primers bind to their target sequences on the single-stranded template.The temperature is then raised to 72C and the Taq DNA polymerase begins to synthesize a new DNA strand complementary to thetemplate strand beginning at the primers. Further cycles of incubation result in the synthesis of increasing amounts of the target sequence.


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target DNA to be copied increases and the reactioncomponents are used up, the increase becomes linearand then reaches a plateau.

Each cycle of DNA synthesis involves three stages(denaturation, primer annealing, elongation), whichtake place at different temperatures and together resultin the synthesis of target DNA.

Denaturation

The reaction is heated to greater than 90C. At thistemperature the double helix is destabilized and theDNAmolecules separate into single strands capable ofbeing copied by the DNA polymerase.

Primer annealing

The reaction is cooled to a temperature that allowsbinding of the primers to the single-stranded DNAwithout permitting the double helix to reform betweenthe template strands. This process is called annealing.The temperature used varies (typically 4565C) and isdetermined by the sequence and the number of bases inthe primers.

Extension

This stage is carried out at the temperature where theDNA polymerase is most active. For Taq, this is 72C.The DNA polymerase, directed by the position of theprimers, copies the intervening target sequence usingthe single-stranded DNA as a template. A total of2040 PCR cycles is carried out depending on theabundance of the target sequence in the templateDNA. Sequences of up to several thousand base pairscan be amplied. To deal with the large number ofseparate incubations needed, the PCR is carried outusing a microprocessor-controlled heating blockknown as a thermal cycler. In the rst cycle, DNAmolecules that extend beyond the target sequence aresynthesized. This is because there is nothing to preventthe DNA polymerase from continuing to copy thetemplate beyond the end of the target sequence. Insubsequent cycles, however, newly synthesized DNAmolecules that end with the primer sequence act astemplates and limit synthesis to the target sequence sothat the amplied DNA contains only the targetsequence.

Variations of the PCR Method

A number of spin-off techniques based on the originalPCRmethod have been developed that have a range ofspecic applications.

Reverse transcriptase PCR

This technique involves using RNA rather than DNAas the template for amplication. The procedure isvery similar to conventional PCR but includes aninitial step in which a DNA copy of the RNA templateis produced using the enzyme reverse transcriptase.This enzyme, which is of viral origin, is a polymeraseand has the unique ability to synthesize DNA from aRNA template. One of the main uses of the reversetranscriptase PCR (RT-PCR) technique is in theanalysis of gene expression. Using gene-specic prim-ers, the presence of an individual messenger RNA(mRNA) species in the total RNA derived from cellsor tissues can be detected. A renement of thetechnique allows the amount of a particular mRNAto be measured. This is known as quantitative RT-PCR and can be used to obtain information on levelsof gene expression.

RT-PCR can also be used to copy the completecoding sequence of a gene. The amplied DNA cansubsequently be used to produce gene clones, whichdirect the synthesis of large amounts of the encodedprotein. By altering the sequence of the cloned DNA,using another PCR-based method known as site-directed mutagenesis, variant proteins with alteredproperties can be produced. This is sometimes referredto as protein engineering.

Degenerate oligonucleotide primer PCR

This technique can be used to amplify related DNAsequences, such as the members of a gene family, or toamplify a DNA sequence from one species based onsequence information from another. The methodinvolves using a mixture of oligonucleotide primersin which alternative nucleotides occur at certainpositions. By incorporating all of the possible basesat a known point of variation, related sequences can beamplied. This technique has been successfully used toidentify new members of gene families such as thehomeobox and cyclin genes.

An extension of the degenerate oligonucleotideprimer PCR (DOP-PCR) technique involves carryingout PCR with primers that are a mixture of completelyrandom sequences. This results in indiscriminateamplication of sequences from throughout thegenome. This technique is referred to as whole-genomePCR and is a useful means of generating manysequences for analysis when only a small amount oftemplate DNA is available, such as in the analysis ofancient DNA or single-cell samples.

Inverse PCR

One of the limitations of conventional PCR is that inorder to design primers it is necessary to know part of


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the DNA sequence to be amplied. However, in somecases only a portion of the sequence of a gene isknown. Inverse PCR is a variation of conventionalPCR that allows unknown sequences anking aknown sequence to be amplied for analysis. Thetechnique involves digesting the template DNA with arestriction enzyme such that the target sequence for agiven PCR will be contained within a larger fragmentcontaining unknown sequence. This fragment can becircularized using the enzyme DNA ligase and theanking regions amplied using the original primerpair facing outwards so that they amplify theremainder of the circle, generating a PCR productcontaining the unknown anking sequence.

Ligase chain reaction

A ligase chain reaction (LCR) technique can be used todetect variations in the DNA sequence of a gene. Twopairs of complementary primers that bind to adjacentpositions on the DNA template are used. The rst twoprimers bind to the template and are joined to eachother by the action of a DNA ligase enzyme. After onedenaturation and annealing cycle, more copies of therst pair anneal to the target DNA and the product ofthe rst round ligation acts as a template for the otherprimer pair. Repeated cycles of denaturation, an-nealing and ligation result in the exponential genera-tion of ligated product. The reaction is very sensitive tothe sequence of the target DNA and if any variation ispresent no ligation product will be produced.

Repetitive element PCR

A signicant portion of the human genome is com-posed of repeated DNA sequences. A well-knownexample is Alu elements, which have an average lengthof 250 bp and occur as almost one million copiesdispersed throughout the genome. Repetitive elementPCR is a method used to amplify interveningsequences between repetitive elements. The methodinvolves the use of a primer that binds a core sequencein the repetitive element. The success of the methodrelies on the repetitive elements occurring in closeproximity and often having opposing orientations.

Applications of the PCR

PCR and its variants are used extensively as a researchtool. Most studies in molecular biology involve theuse of PCR at some stage, normally as part of anoverall strategy and in association with other tech-niques. For example, DNA amplied by PCR can beused for DNA sequencing, as a probe in Northern andSouthern blotting, and to generate clones.

PCR has applications in many areas of research inbiology and medicine as well as in unexpected subjectssuch as anthropology and archeology. It is also animportant techniqueusedinthebiotechnologyindustry.PCR has made important contributions to research inmany areas, some of which are described below.

Inherited diseases

These disorders are caused by gene mutations passedon from parents to their children. Examples includehemophilia and cystic brosis. PCR is used to amplifygene sequences, which can then be screened for disease-causing mutations. The information obtained hasdramatically improved our understanding of thesedisorders and has produced the important additionalbenet of allowing carriers of the disorders to beidentied.

Cancer research

PCRhas been widely used in studies of the role of genesin cancer. For example, mutations in oncogenes andtumor-supressor genes have been identied in DNAfrom tumors using PCR-based strategies. This hasimproved our understanding of how cancer develops.

Forensic science

By amplifying repetitive sequences, PCR can be usedto identify individuals from samples of their DNA.This can be used, for example, to link individuals withforensic DNA samples from the scene of a crime.Analysis of variable sequences is also used in tissuetyping to match organ donors with recipients and inanthropology to study the origins of races of people.

Biotechnology

PCR has played an important role in the production ofrecombinant proteins such as insulin and growthhormone, which are widely used as drugs, and in thedevelopment of recombinant vaccines such as that forthe hepatitis B virus.

See alsoPolymerase Chain Reaction (PCR): Design and Optimization of

Reactions

Further Reading

Erlich HA (ed.) (1989) PCR Technology: Principles and Applicationsfor DNA Amplification. New York, NY: Stockton Press.

Erlich HA and Arnheim N (1992) Genetic analysis using the poly-merase chain reaction. Annual Review of Genetics 26: 479506.

McPherson MP and Hames BD (eds.) (1995) PCR 2: A PracticalApproach. Oxford, UK: IRL Press.

Nanda SK and Jain SK (1995) In vitro nucleic acid amplicationsystems. Current Science 66: 421429.


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Ohan NW and Heikkila JJ (1995) Reverse transcription polymerasechain reaction an overview of the technique and its applica-tions. Biotechnology Advances 11: 1329.

Reischl U and Kochanowski B (1995) Quantitative PCR a surveyof the present technology. Molecular Biotechnology 3: 5571.

Twyman RM (1998) Advanced Molecular Biology: A ConciseReference, pp. 279285. Oxford, UK: Bios Scientic Publishers.

Winter PC, Hickey GI and Fletcher HL (2002) Instant Notesin Genetics, pp. 255258. Oxford, UK: Bios ScienticPublishers.


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