PCR is a technique that utilizes DNA replication to make many copies of a desire
ID: 69126 • Letter: P
Question
PCR is a technique that utilizes DNA replication to make many copies of a desired sequence of DNA from as little as one source molecule.
Why do we use Taq DNA polymerase instead of human DNA polymerase in PCR relations?
What are the properties of the DNA primers that you, as a scientist, would add to a PCR reaction to ensure it is successful? (Include discussion of polarity (5’-3’ or 3’-5’), which strand each primer bind to, complementary and anything else you think is necessary). Remember that the enzyme being used is a DNA polymerase, so think of the properties of DNA polymerases when discussing primers.
Explanation / Answer
The PCR technique is carried out by three different steps namely Denaturation, Annealing and extension.
The denaturation of the DNA strands occurs at temperature more than 90oC, so the normally DNA polymerase cannot withstand temperatures more than 40oC. Hence usage of Taq polymerase is employed in the PCR technique. Taq polymerase can stable upto 95oC. It is isolated from a thermophile Thermus aqaticus.
The annealing step is a process where the forward and reverse primers bind to the denatured strands. So the melting temperature of the DNA primers should be equal to +/- 5oC of the annealing temperature. DNA primers should have 50-60% of GC content. The forward primer is exactly same as that of + strand of DNA and will bind to - strand of DNA as it is complimentary. The reverse primer will be complement to - strand and will bind to the + strand at the 3' end.
As DNA polymerase can extend the primers in 5' - 3' direction.
5'-----------------------------------------------------------------------------------------3'
3' <----------------5' Forward primer
5'-------------->3' Reverse Primer
3'-----------------------------------------------------------------------------------------5'
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