Polymerase chain reaction (PCR) 8/1/03. By Richard Twyman The polymerase chain reaction (PCR) is a widely used technique for the selective amplification of particular DNA sequences, such as individual genes.

The PCR is quick and sensitive and robust and is particularly useful when dealing with small amounts of DNA, or where rapid and high-throughput screening is required.

How does it work?

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1 of 3 The polymerase chain reaction (PCR) is used to make huge numbers of copies of a piece of DNA. The reaction begins by heating up the DNA template to 94°C, which splits (denatures) the double strands into single strands. The sample is then cooled to about 54°C, which allows the primers to stick (anneal) to the template. When the sample is heated up again to 72°C, the polymerase enzyme uses the primers as starting points to copy the single strands.

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2 of 3 The cycle of denaturation, primer annealing and primer extension is repeated over and over again (using a machine that automates the heating and cooling of the samples), each time producing more copies of the original template.

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3 of 3 During repeated rounds of these reactions, the number of newly synthesized DNA strands increases exponentially. After 25 to 30 cycles, the initial template DNA will have been copied several million-fold.

In normal DNA synthesis reactions, a single primer sticks to a single-stranded DNA template and is extended by DNA polymerase in the presence of the four nucleotides (the building blocks of DNA). The product accumulates in a linear fashion because the amount of template remains the same throughout the reaction.

The PCR is different because two primers are used. These flank the region to be amplified and face inwards, on opposite DNA strands, so that synthesis occurs across the central region. The products of the DNA synthesis reaction can themselves act as templates for DNA synthesis. Therefore, after one round of synthesis, the amount of template DNA is doubled.

Doubling occurs in every cycle of the PCR leading to exponential amplification of the target. After 25 cycles there are over 8 000 000 copies!

Each time DNA synthesis is carried out, a single stranded template is converted into a double-stranded product. Before further synthesis is possible, this product must be separated into single strands.

Each PCR cycle therefore comprises three processes: denaturation (the separation of DNA strands), primer annealing and primer extension. Denaturation is achieved by heating the DNA while primer annealing and primer extension occur at lower temperatures. The PCR therefore involves temperature cycling, and the DNA polymerase used in the reaction must be tolerant of high temperatures and rapid heating and cooling.

How is it used?

The PCR is useful where the amount of starting material is limited or poorly preserved. Examples of PCR applications include cloning DNA from single cells, prenatal screening for mutations in early human embryos, and the forensic analysis of DNA sequences in samples such as fingerprints, blood stains, semen or hairs.

The PCR is also very useful where many samples have to be processed in parallel. For example, the large-scale analysis of single nucleotide polymorphisms involves PCR-based techniques.

A modification called reverse transcriptase PCR (RT-PCR) allows RNA sequences to be amplified indirectly. The enzyme reverse transcriptase is used to copy the RNA into complementary DNA (cDNA); the cDNA is then amplified by PCR. This technique is useful for analysing gene expression.