Polymerase chain reaction

"PCR" redirects here. For other uses, see PCR (disambiguation).
A strip of eight PCR tubes, each containing a 100 μl reaction mixture

Polymerase chain reaction (PCR) is a technique used in molecular biology to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. It is an easy and cheap tool to amplify a focused segment of DNA, useful for such purposes as the diagnosis and monitoring of genetic diseases, identification of criminals (in the field of forensics), and studying the function of a targeted segment of DNA.[1]

Developed in 1983 by Kary Mullis,[2][3] PCR is now a common and often indispensable technique used in clinical laboratories and research laboratories for a variety of applications.[4][5] These include DNA cloning for sequencing, DNA-based phylogeny, or functional analysis of genes; the diagnosis of hereditary diseases; the identification of genetic fingerprints (used in forensic sciences and DNA paternity testing); and the detection of pathogens in nucleic acid tests for the diagnosis of infectious diseases. In 1993, Mullis was awarded the Nobel Prize in Chemistry along with Michael Smith for his work on PCR.[6]

The method relies on thermal cycling, consisting of cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA. Primers (short DNA fragments) containing sequences complementary to the target region along with a DNA polymerase, which the method is named after, are key components to enable selective and repeated amplification. As PCR progresses, the DNA generated is itself used as a template for replication, setting in motion a chain reaction in which the DNA template is exponentially amplified. PCR can be extensively modified to perform a wide array of genetic manipulations. PCR is not generally considered to be a recombinant DNA method, as it does not involve cutting and pasting DNA, only amplification of existing sequences.

Almost all PCR applications employ a heat-stable DNA polymerase, such as Taq polymerase (an enzyme originally isolated from the bacterium Thermus aquaticus). This DNA polymerase enzymatically assembles a new DNA strand from DNA building-blocks, the nucleotides, by using single-stranded DNA as a template and DNA oligonucleotides (also called DNA primers), which are required for initiation of DNA synthesis. The vast majority of PCR methods use thermal cycling, i.e., alternately heating and cooling the PCR sample through a defined series of temperature steps.

In the first step, the two strands of the DNA double helix are physically separated at a high temperature in a process called DNA melting. In the second step, the temperature is lowered and the two DNA strands become templates for DNA polymerase to selectively amplify the target DNA. The selectivity of PCR results from the use of primers that are complementary to the DNA region targeted for amplification under specific thermal cycling conditions.

Placing a strip of eight PCR tubes into a thermal cycler


A thermal cycler for PCR
An older model three-temperature thermal cycler for PCR

PCR amplifies a specific region of a DNA strand (the DNA target). Most PCR methods typically amplify DNA fragments of between 0.1 and 10 kilo base pairs (kbp), although some techniques allow for amplification of fragments up to 40 kbp in size.[7] The amount of amplified product is determined by the available substrates in the reaction, which become limiting as the reaction progresses.[8]

A basic PCR set up requires several components and reagents.[9] These components include:

The PCR is commonly carried out in a reaction volume of 10–200 μl in small reaction tubes (0.2–0.5 ml volumes) in a thermal cycler. The thermal cycler heats and cools the reaction tubes to achieve the temperatures required at each step of the reaction (see below). Many modern thermal cyclers make use of the Peltier effect, which permits both heating and cooling of the block holding the PCR tubes simply by reversing the electric current. Thin-walled reaction tubes permit favorable thermal conductivity to allow for rapid thermal equilibration. Most thermal cyclers have heated lids to prevent condensation at the top of the reaction tube. Older thermocyclers lacking a heated lid require a layer of oil on top of the reaction mixture or a ball of wax inside the tube.


Typically, PCR consists of a series of 20–40 repeated temperature changes, called cycles, with each cycle commonly consisting of 2–3 discrete temperature steps, usually three (Figure below). The cycling is often preceded by a single temperature step at a high temperature (>90 °C), and followed by one hold at the end for final product extension or brief storage. The temperatures used and the length of time they are applied in each cycle depend on a variety of parameters. These include the enzyme used for DNA synthesis, the concentration of divalent ions and dNTPs in the reaction, and the melting temperature (Tm) of the primers.[12]

The processes of denaturation, annealing and elongation constitute of one cycle. Multiple cycles are required to amplify DNA. The formula used to calculate the number of DNA copies formed after a given # of cycles repeated is, 2^# of cycles.[16]

Ethidium bromide-stained PCR products after gel electrophoresis. Two sets of primers were used to amplify a target sequence from three different tissue samples. No amplification is present in sample #1; DNA bands in sample #2 and #3 indicate successful amplification of the target sequence. The gel also shows a positive control, and a DNA ladder containing DNA fragments of defined length for sizing the bands in the experimental PCRs.

To check whether the PCR generated the anticipated DNA fragment (also sometimes referred to as the amplimer or amplicon), agarose gel electrophoresis is employed for size separation of the PCR products. The size(s) of PCR products is determined by comparison with a DNA ladder (a molecular weight marker), which contains DNA fragments of known size, run on the gel alongside the PCR products (see Fig. 3).


The PCR process can be divided into three stages:

Exponential amplification: At every cycle, the amount of product is doubled (assuming 100% reaction efficiency). The reaction is very sensitive: only minute quantities of DNA must be present.[17]

Leveling off stage: The reaction slows as the DNA polymerase loses activity and as consumption of reagents such as dNTPs and primers causes them to become limiting.

Plateau: No more product accumulates due to exhaustion of reagents and enzyme.


Main article: PCR optimization

In practice, PCR can fail for various reasons, in part due to its sensitivity to contamination causing amplification of spurious DNA products. Because of this, a number of techniques and procedures have been developed for optimizing PCR conditions.[18][19] Contamination with extraneous DNA is addressed with lab protocols and procedures that separate pre-PCR mixtures from potential DNA contaminants.[9] This usually involves spatial separation of PCR-setup areas from areas for analysis or purification of PCR products, use of disposable plasticware, and thoroughly cleaning the work surface between reaction setups. Primer-design techniques are important in improving PCR product yield and in avoiding the formation of spurious products, and the usage of alternate buffer components or polymerase enzymes can help with amplification of long or otherwise problematic regions of DNA. Addition of reagents, such as formamide, in buffer systems may increase the specificity and yield of PCR.[20] Computer simulations of theoretical PCR results (Electronic PCR) may be performed to assist in primer design.[21]


Selective DNA isolation

PCR allows isolation of DNA fragments from genomic DNA by selective amplification of a specific region of DNA. This use of PCR augments many methods, such as generating hybridization probes for Southern or northern hybridization and DNA cloning, which require larger amounts of DNA, representing a specific DNA region. PCR supplies these techniques with high amounts of pure DNA, enabling analysis of DNA samples even from very small amounts of starting material.

Other applications of PCR include DNA sequencing to determine unknown PCR-amplified sequences in which one of the amplification primers may be used in Sanger sequencing, isolation of a DNA sequence to expedite recombinant DNA technologies involving the insertion of a DNA sequence into a plasmid, phage, or cosmid (depending on size) or the genetic material of another organism. Bacterial colonies (such as E. coli) can be rapidly screened by PCR for correct DNA vector constructs.[22] PCR may also be used for genetic fingerprinting; a forensic technique used to identify a person or organism by comparing experimental DNAs through different PCR-based methods.

Some PCR 'fingerprints' methods have high discriminative power and can be used to identify genetic relationships between individuals, such as parent-child or between siblings, and are used in paternity testing (Fig. 4). This technique may also be used to determine evolutionary relationships among organisms when certain molecular clocks are used (i.e., the 16S rRNA and recA genes of microorganisms).[23]

Electrophoresis of PCR-amplified DNA fragments. (1) Father. (2) Child. (3) Mother. The child has inherited some, but not all of the fingerprint of each of its parents, giving it a new, unique fingerprint.

Amplification and quantification of DNA

Because PCR amplifies the regions of DNA that it targets, PCR can be used to analyze extremely small amounts of sample. This is often critical for forensic analysis, when only a trace amount of DNA is available as evidence. PCR may also be used in the analysis of ancient DNA that is tens of thousands of years old. These PCR-based techniques have been successfully used on animals, such as a forty-thousand-year-old mammoth, and also on human DNA, in applications ranging from the analysis of Egyptian mummies to the identification of a Russian tsar and the body of English king Richard III.[24]

Quantitative PCR methods allow the estimation of the amount of a given sequence present in a sample—a technique often applied to quantitatively determine levels of gene expression. Quantitative PCR is an established tool for DNA quantification that measures the accumulation of DNA product after each round of PCR amplification.

Medical applications

PCR has been applied to a large number of medical procedures:

Infectious disease applications

PCR allows for rapid and highly specific diagnosis of infectious diseases, including those caused by bacteria or viruses.[27] PCR also permits identification of non-cultivatable or slow-growing microorganisms such as mycobacteria, anaerobic bacteria, or viruses from tissue culture assays and animal models. The basis for PCR diagnostic applications in microbiology is the detection of infectious agents and the discrimination of non-pathogenic from pathogenic strains by virtue of specific genes.[27][28]

Characterization and detection of infectious disease organisms have been revolutionized by PCR in the follow ways:

Forensic applications

The development of PCR-based genetic (or DNA) fingerprinting protocols has seen widespread application in forensics:

Research applications

PCR has been applied to many areas of research in molecular genetics:


DNA polymerase is prone to error, which in turn causes mutations in the PCR fragments that are made. Additionally, the specificity of the PCR fragments can mutate to the template DNA, due to nonspecific binding of primers. Furthermore, prior information on the sequence is necessary in order to generate the primers.[31]


Main article: Variants of PCR


Diagrammatic representation of an example primer pair. The use of primers in an in vitro assay to allow DNA synthesis was a major innovation that allowed the development of PCR.

A 1971 paper in the Journal of Molecular Biology by Kjell Kleppe and co-workers in the laboratory of H. Gobind Khorana first described a method using an enzymatic assay to replicate a short DNA template with primers in vitro.[61] However, this early manifestation of the basic PCR principle did not receive much attention at the time, and the invention of the polymerase chain reaction in 1983 is generally credited to Kary Mullis.[62]

"Baby Blue", a 1986 prototype machine for doing PCR

When Mullis developed the PCR in 1983, he was working in Emeryville, California for Cetus Corporation, one of the first biotechnology companies. There, he was responsible for synthesizing short chains of DNA. Mullis has written that he conceived of PCR while cruising along the Pacific Coast Highway one night in his car.[63] He was playing in his mind with a new way of analyzing changes (mutations) in DNA when he realized that he had instead invented a method of amplifying any DNA region through repeated cycles of duplication driven by DNA polymerase. In Scientific American, Mullis summarized the procedure: "Beginning with a single molecule of the genetic material DNA, the PCR can generate 100 billion similar molecules in an afternoon. The reaction is easy to execute. It requires no more than a test tube, a few simple reagents, and a source of heat."[64] He was awarded the Nobel Prize in Chemistry in 1993 for his invention,[6] seven years after he and his colleagues at Cetus first put his proposal to practice. However, some controversies have remained about the intellectual and practical contributions of other scientists to Mullis' work, and whether he had been the sole inventor of the PCR principle (see below).

At the core of the PCR method is the use of a suitable DNA polymerase able to withstand the high temperatures of >90 °C (194 °F) required for separation of the two DNA strands in the DNA double helix after each replication cycle. The DNA polymerases initially employed for in vitro experiments presaging PCR were unable to withstand these high temperatures.[4] So the early procedures for DNA replication were very inefficient and time-consuming, and required large amounts of DNA polymerase and continuous handling throughout the process.

The discovery in 1976 of Taq polymerase — a DNA polymerase purified from the thermophilic bacterium, Thermus aquaticus, which naturally lives in hot (50 to 80 °C (122 to 176 °F)) environments[14] such as hot springs — paved the way for dramatic improvements of the PCR method. The DNA polymerase isolated from T. aquaticus is stable at high temperatures remaining active even after DNA denaturation,[15] thus obviating the need to add new DNA polymerase after each cycle.[5] This allowed an automated thermocycler-based process for DNA amplification.

Patent disputes

The PCR technique was patented by Kary Mullis and assigned to Cetus Corporation, where Mullis worked when he invented the technique in 1983. The Taq polymerase enzyme was also covered by patents. There have been several high-profile lawsuits related to the technique, including an unsuccessful lawsuit brought by DuPont. The pharmaceutical company Hoffmann-La Roche purchased the rights to the patents in 1992 and currently holds those that are still protected.

A related patent battle over the Taq polymerase enzyme is still ongoing in several jurisdictions around the world between Roche and Promega. The legal arguments have extended beyond the lives of the original PCR and Taq polymerase patents, which expired on March 28, 2005.[65]


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