In answer section.
- The polymerase chain reaction (PCR) is a process to 'amplify' DNA to provide sufficient quantities for further analysis.
PCR involves repeated cycles of heating and cooling of the reaction mixture. A single cycle is as follows:
95 degrees Celsius for 30s55 degrees Celsius for 30s72 degrees Celsius for 90s
a) The most commonly used DNA polymerase in PCR is the polymerase from the bacterium Thermophilius acquaticus. Why is human DNA polymerase not frequently used? [2]
b) Other than the DNA polymerase and the DNA to be amplified, identify two other reactants present in the reaction mixture. [2]
c) Why is the reaction mixture heated to 95 degrees Celsius initially? [1]
d) Once the DNA has been amplified, it has a variety of uses. Identify and describe two potential uses of the DNA. [2]
- a) Human DNA polymerase would be denatured at 95 degrees, whereas Taq polymerase is thermostable. If human DNA polymerase was used, fresh enzyme would have to be added in every cycle, which would be time consuming and wasteful.
b) - Two single-stranded DNA primers, complementary to the 3' end of each of the DNA strands to be amplified.
- dNTPs to be added to the DNA primers during elongation by the DNA polymerase.
c) To break the hydrogen bonds holding the two DNA strands together. This provides a binding site for the primers, which the DNA polymerase can then elongate.
d) 1) Small amounts of DNA can be recovered from crime scenes. This can be amplified using PCR to be compared to national forensic databases.
- Certain genetic alleles can indicate a predisposition to diseases, such as cancer. In instances where there is a family history of disease, descendents may elect to undergo genetic testing to determine whether they carry the gene with the associated risk.
