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Polymerase chain reaction

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The polymerase chain reaction (PCR) is a diagnostic method and is a type of nucleic acid amplification technique frequently used in medicine, molecular biology, and law enforcement, as well as in agriculture and food quality and safety control, to amplify the amount of DNA present in a sample in order to detect it. Detection of DNA can help in "detection of difficult-to-isolate pathogens, mutation analysis, genetic testing, DNA sequencing, and analyzing evolutionary relationships."[1]

Typically, the reaction is repeated a few dozen times, during which the DNA content is doubled at each cycle. The technique requires the DNA to be amplified, a DNA poylmerase enzyme, such as Taq-polymerase, to elongate primers, two DNA primer sequences that are complimentary to the ends of the sample DNA, and DNA nucleotides to be incorporated into the new strands of DNA.

Process

A PCR reaction is a nucleic acid amplification technique and consists of three stages: denaturation, annealing and extension. This cycle is repeated until enough DNA is produced, generally after 30-40 cycles.

Denaturation

In the first step, the denaturation, the temperature is raised to above 90° C. Due to the high temperature, the hydrogen bonds between the two strands of DNA break and the strands are separated. Now the desired target DNA is made accessible for the next stage.

Annealing

In the next stage, the annealing, the temperature is cooled down between 40 and 60° C. The precise temperature is very important and should be determined before every reaction. After it is cooled down, the primers will bind to the DNA. Primers are short single strands of DNA that are complementary to the 3’ end of the target DNA.

Extension

In the third stage, the extension, the temperature is raised again, most of the time to 72° C, the optimal temperature of Taq-polymerase. The polymerase will bind to the primers and copy the target DNA in one direction, to the 5’ end. When the polymerase is done, two partially double-stranded DNA molecules are produced.

References

  1. Anonymous (2017), Polymerase chain reaction (English). Medical Subject Headings. U.S. National Library of Medicine.