The Amplification of DNA by Polymerase Chain Reaction
Introduction: Polymerase chain reaction (PCR) is a technique for copying a piece of DNA a billion-fold. As the name suggests, the process creates a chain of many pieces, in this case the pieces are nucleotides, and the chain is a strand of DNA. PCR is an enzyme-mediated reaction, and as with any enzyme, the reaction must occur at the enzyme's ideal operating temperature. The enzymes that are used for the PCR are DNA-dependent DNA polymerases (DDDP) resulting from thermophilic bacteria. The enzymes function at higher temperatures than the enzymes we commonly use in the laboratory or have working in our bodies. These DNA polymerases operate at 60-75°C, and can even survive at temperatures above 90°C. This is important because a part of the PCR requires that the reaction reaches approximately 94°C. Apart from the DNA polymerase, PCR needs a DNA template to copy, and a pair of short DNA sequences called oligonucleotides or "primers" to get the DNA polymerase started. Generally speaking, there are three steps identified by incubating at different temperatures. These three steps make up a PCR cycle. The first step of the PCR cycle is known as denaturation. At temperatures above 90°C, the double-stranded DNA denatures. That means the weak hydrogen bonds that usually hold the two complementary strands together at normal temperatures are disrupted resulting in two single stranded DNA strands. The second step is known as annealing. At the annealing temperature, which was 37°C in our experiment, primers that collide with their complementary sequence can bind to it. The chance of such an encounter happening is increased because we use an excess amount of each primer in the reaction mixture compared to the number of template molecules present. The final step of the PCR cycle is known as extension. At the extension temperature, which was 72°C in our experiment, the DNA polymerase binds to the hybridized primer and begins...
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