Explain the Polymerase Chain Reaction (PCR) as a technique to make copies of DNA.

PCR takes place in three stages - denaturation, annealing, and extension. The process begins with a very small quantity of DNA being loaded into a PCR machine. The hydrogen bonds that exist between the two strands of DNA can be broken and the strands separated if the DNA is heated to a high temperature. If the strands are cooled again, then hydrogen bonds can form again - a process is called re-annealing. The PCR machine hence separates the DNA strands by heating them to 95 degrees Celsius for 15 seconds, then cooling quickly to 54 degrees Celcius. This allows the re-annealing of parent strands to form double-stranded DNA. There are also short sections of single-stranded DNA called primers present, which bind to target sequences. Since a large number of primers are present, they prevent the re-annealing of the parent strands. Copying of the single parent strands then starts from the primers.The next stage of PCR involves the synthesis of double- stranded DNA, using the single strands with primers as templates. The enzyme Taq polymerase is used to do this - originally obtained from Thermus aquaticus in hot springs, so this bacterium and its DNA polymerase are adapted to be very heat-stable to resist denaturation. Hence, Taq polymerase is used as it can resist the brief period at 95 °C used to separate the DNA strands. It would work at the lower temperature of 54 °C that is used to attach the primers, but has an optimum temperature is 72 °C and so the reaction mixture is heated to this temperature for the period when Taq DNA polymerase is working. At this optimum temperature a rapid rate of DNA replication takes place, and once enough time has elapsed for replication of the selected base sequence to be complete, the next cycle is started by heating to 95 °C. A cycle of PCR can be completed in less than two minutes and in less than an hour, thirty cycles can be completed to amplify the DNA by a factor of a billion.

Answered by Aashna B. Biology tutor


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