Polymerase chain reaction

The Polymerase Chain Reaction (PCR) is a biochemical technology in molecular biology that amplifies a single or few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence.

COLD-PCR applications

Procedure

The PCR technique is used in molecular biology to make several copies of a specific DNA segment. Using PCR, a single copy (or more) of a DNA sequence is exponentially amplified to generate thousands to millions of more copies of that particular DNA segment. PCR is now a common and often indispensable technique used in medical laboratory and clinical laboratory research for a broad variety of applications. 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 paternity testing); and the detection and diagnosis of infectious diseases.

Polymerase chain reaction diagram

PCR follows three steps, which are repeated for 20 to 40 cycles. This cycling is often preceded by a single temperature step (called hold) at a high temperature (>90°C), and followed by one hold at the end for final product extension or brief storage.

• Denaturation at 94–96°C: During the denaturation, the double strand melts open to single-stranded DNA, all enzymatic reactions stop (for example, the extension from a previous cycle).
• Annealing at ~68°C: The primers are jiggling around, caused by the Brownian motion. Ionic bonds are constantly formed and broken between the single-stranded primer and the single-stranded template. The more stable bonds last a little bit longer (primers that fit exactly) and on that little piece of double-stranded DNA (template and primer), the polymerase can attach and starts copying the template. Once there are a few bases built in, the ionic bond is so strong between the template and the primer, that it does not break anymore.
• Elongation at 72°C: The DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template in 5' to 3' direction, condensing the 5'-phosphate group of the dNTPs with the 3'-hydroxyl group at the end of the nascent (extending) DNA strand.

Variations of PCR

Several variations of PCR have been developed for different applications.

• Reverse transcription PCR (RT-PCR): This is used for amplifying DNA from RNA. Reverse transcriptase reverse transcribes RNA into cDNA, which is then amplified.
• Real-time PCR: Also known as quantitative PCR (qPCR), it monitors the amplification of a targeted DNA molecule during the PCR, i.e., in real-time, and not at its end, as in conventional PCR. Real-time PCR can be used quantitatively and semi-quantitatively, such as in a comparative Cq (quantification cycle) value relative to the amount of DNA input or using absolute quantification.
• Multiplex-PCR: Multiplex-PCR consists of multiple primer sets within a single PCR mixture to produce amplicons of varying sizes that are specific to different DNA sequences. By targeting multiple genes at once, additional information may be gained from a single test run that otherwise would require several times the reagents and more time to perform.
• Hot-start PCR: A technique performed manually by heating the reaction components to the DNA melting temperature (e.g., 95°C) before adding the polymerase.
• Assembly PCR: Assembly PCR or Polymerase Cycling Assembly (PCA) is the synthesis of long DNA structures by performing PCR on a pool of long oligonucleotides with short overlapping segments. The oligonucleotides alternate between sense and antisense directions, and the overlapping segments determine the order of the PCR fragments, thereby selectively producing the final long DNA product.

Applications of PCR

PCR has a wide range of applications in different areas of biology and medicine, including:

• Molecular biology: PCR is the cornerstone of modern molecular biology.
• Medical and diagnostic applications: PCR allows early diagnosis of malignant diseases such as leukemia and lymphomas, which is currently the highest developed in cancer diagnosis and in forensic medicine.
• Genetic testing: PCR is used to analyze genes associated with genetic disorders, including mutations in oncogenes and tumor suppressor genes in cancer.
• Infectious diseases: PCR allows for the identification of infectious diseases, such as HIV, HPV, Chlamydia trachomatis, and Neisseria gonorrhoeae.
• Forensic sciences: PCR is used in forensic sciences to identify individuals or animals by their DNA profiles.

See also

• DNA sequencing
• Real-time polymerase chain reaction
• Reverse transcription polymerase chain reaction
• Taq polymerase

External links

National Institutes of Health: Polymerase Chain Reaction (PCR) Khan Academy: Polymerase Chain Reaction (PCR)

Further Reading

Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., ... & Erlich, H. A. (1988). Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science, 239(4839), 487-491. Bustin, S. A., & Nolan, T. (2017). Talking the talk, but not walking the walk: RT-qPCR as a paradigm for the lack of reproducibility in molecular research. European Journal of Clinical Investigation, 47(10), 756-774.

Polymerase chain reaction Resources
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