Polymerase chain reaction (PCR) is a technique used to amplify specific regions of DNA for further analysis.
PCR can start with a small amount of genomic DNA and make billions of copies of the desired segment of DNA in a few hours. To achieve this, researchers prepare a reaction mixture of DNA containing the target sequence, heat-stable DNA polymerase, primers—short pieces of DNA complementary to each end of the target sequence (primers will ideally have a high content of G and C bases at the 5′ and 3′ ends as they form stronger hydrogen bonds) —, and deoxynucleotides.
The reaction occurs in three-step cycles. In the first step, high temperature denatures DNA (i.e. strand separation). That is why heat-stable polymerase is an essential requirement for PCR. For this purpose, scientists isolated Taq polymerase from the thermostable bacterium Thermus aquaticus. This organism lives in hot springs with high temperatures used in PCR.
In the second step, the temperature of the reaction mixture cools down so that primers bind (i.e. form hydrogen bonds with) their target sequence on DNA. Lastly, DNA polymerase extends both primers via attaching nucleotides to 3’ end of the primer. Repeating this cycle many times increase the proportion of the target DNA sequence in the reaction mixture exponentially.
PCR serves for many purposes in laboratories, such as the cloning of gene fragments to analyze genetic diseases, the identification of contaminant foreign DNA in a sample, and the amplification of DNA from ancient samples for sequencing.
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Key Points
• PCR is a regular technique used in genetics to amplify the target DNA sequence.
• It has many applications from forensics to diagnosing infectious diseases.
• The reaction occurs in three-step cycles. In the first step, high temperature denatures DNA (i.e. strand separation).
• In the second step, the temperature of the reaction mixture cools down so that primers bind
• Lastly, DNA polymerase extends both primers via attaching nucleotides to 3’ end of the primer.
Key Terms
Deoxynucleotide: a building block molecule of DNA.
DNA polymerase: an enzyme that produces DNA molecules from its building blocks – deoxynucleotides.
Gene cloning: a biological technique that creates many identical copies of a gene (or any other DNA fragment) and directs its replication within a host organism
Primer: a short nucleic acid sequence that provides a starting point for DNA synthesis
Hydrogen bond: an attractive interaction between a hydrogen atom from a molecule or a molecular fragment X–H in which X is more electronegative than H
3‘ end: nucleic acid strand which terminates at the hydroxyl (-OH) chemical group attached to the third carbon in the sugar-ring
Taq polymerase: is a thermostable DNA polymerase