BME100 f2014:Group2 L4
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PCR - The Underlying Technology
There are several components of a PCR reaction that serve their own specific function. These components include template DNA, primers, taq polymerase, and deoxyribonucleotides. The function of extracted DNA from the cells is to serve as the template DNA from which the copies of DNA are made. This is important since the purpose of PCR is to make many DNA copies. The function of primers is to attach to a specific site of DNA in order to copy the desired DNA sequence. The primers have little chance of targeting the wrong site, so it is useful to successfully copy a desired DNA sequence. The function of the taq polymerase is to synthesise a new strand of DNA complementary to the starting template strand. The deoxyribonucleotides are the A’s, C’s, G’s, and T’s that make up DNA. Deoxyribonucleotides serve as the genetic building blocks that are used to create the DNA copies in a PCR reaction.
Steps of Thermal Cycling
During the initial step of thermal cycling which occurs at ninety five degrees Celsius for three minutes, the taq polymerase is being activated by the heat. It is heated to near boiling to allow the DNA to denature. During the denature step at ninety five degrees Celsius for thirty seconds, the double stranded DNA template denatures, so it is now single stranded DNA. Next, during the anneal step at fifty seven degrees Celsius for thirty seconds, short DNA sequences known as primers bind to, or anneal to, complementary matches on the single stranded DNA template. During the extend step at seventy two degrees Celsius for thirty seconds, the taq polymerase performs synthesis, it attaches nucleotides to the correct place along the single stranded DNA template so that a new complementary strand of DNA is extended from the primer. During the final step at seventy two degrees Celsius for three minutes, the taq polymerase ensures that any remaining single-stranded DNA is fully extended. During the final hold, there is a short term storage of the reaction.
Base Pairing and Thermal Cycling
The base adenine anneals to the base thymine. The base themine anneals to the base adenine. The base cytosine anneals to the base guanine. The base guanine anneals to the base cytosine. Base pairing occurs during two steps of thermal cycling, anneal and extend. During anneal, primers bind to complementary matches on the single stranded DNA template. During the extend step, the taq polymerase performs synthesis. The taq polymerase attaches nucleotides to the correct place along the single stranded DNA template to form a new complementary strand of DNA.