Polymerase Chain Reaction (PCR)
Background to Polymerase Chain Reaction
Each eukaryotic cell usually have two copies of each gene, and prokaryotic cells have one copy. This small amount of DNA poses an issue for scientists who want to work with it. Or alternatively, only a small amount of DNA may be available to analyse, for example from a crime scene or in DNA samples obtained from bones.
PCR is a biotechnological technique which aids to fix this problem. Taq polymerase is an enzyme derived from the bacterium Thermus acquaticus which originates in hot springs, living in temperatures up to 95ºC. Most enzymes would denature at this temperature, but Taq remains stable, hence its use in PCR. Taq is similar to DNA polymerase in that it catalyses the formation of new strands of DNA.
What is Polymerase Chain Reaction?
PCR, polymerase chain reaction, is a cyclic method used to rapidly amplify relatively small amounts of particular sequences of DNA into large numbers of copies. The amplified DNA has different uses based on the scenario. The process involves a series of temperature cycles which is automatically controlled by machines called thermal cyclers or thermocyclers, which provide tight control over both reaction temperature and the duration of each temperature step. As each cycle doubles the number of DNA strands, billions of copies of the DNA sample can be made just within a few hours. Ten cycles of PCR produces 1,024 copies (2^10), twenty cycles produces 1,048,576 (2^20) and thirty cycles produces 1,073,741,824 (2^30)!
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What are the requirements for PCR?
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The DNA to be copied (or the template DNA strand)
A buffer solution (contains salts/chemical to maintain correct pH for Taq polymerase)
A supply of the four nucleotide bases (dNTPs)
Two sets of DNA primers (short single-stranded DNA sequences)
What is the process of PCR?
Image: Biotechnology process image, Image by Enzoklop, Sourced Under a Creative Commons 4.0 License from Wiki Commons.
Denaturation: The double stranded DNA is heated to 95ºC which breaks the weak hydrogen bonds holding the complementary strands together. The template strands have free, exposed nucleotide bases which will be used in the synthesis of the new DNA strands.
Annealing: The temperature is reduced to 50–60ºC which allows the DNA primers to anneal to complementary sequences on opposite ends of each strand. The DNA used is either genomic (from the original DNA) or PCR products from the last cycle.
Extension: The temperature is raised to 72ºC which is the optimum temperature for Taq polymerase. Starting from the primers, new DNA strands are synthesised using Taq polymerase and the available free nucleotides (dNTPs). There are now two copies of each strand (twice the amount).
What are the applications of PCR?
The amplified amount of DNA is suitable for many uses, such as in gel electrophoresis, DNA profiling, creating a DNA fingerprint etc. In terms of specific scenarios, this may be for crime scene analysis, or in some cases, maternity/paternity testing.