Question

1. If you started a PCR protocol with 10 double-stranded molecules of DNA, how many double-stranded molecules of the product would you have after 5 cycles of PCR?

2. What is the purpose of using DNA polymerase from Thermus aquaticus for the polymerase chain reaction rather than a DNA polymerase from a better characterized bacterium such as E. coli?

Answer 1

1. The number of double-stranded DNA pieces is doubled in each cycle of PCR. If a PCR starts with 1 molecule of ds DNA the after one cycle they will be 2. after 2 cycles they will be 4, so that after n cycles the product will be 2^n. A single molecule of ds DNA after 5 cycles produce 2⁵ = 32 molecules of DNA. so, 10 ds DNA after 5 cycles will produce 10 X 32 =320 molecule of ds DNA.

2. The polymerase reaction (Extension) is carried out in the 72^oC temperature to avoid nonspecific amplification. polymerase enzyme is a necessary enzyme for the amplification of the DNA molecule and the function of this enzyme is temperature specific.  The polymerase enzyme of E.coli shows optimal activity at 37°C. But in PCR the amplification must be carried out in much higher temperature (72^oC). Therefore the polymerase of the E.coli can not be used in PCR. The bacteria T. aquaticus lives in hot springs and hydrothermal vents (temperature tolerance) and the DNA polymerase is called Taq polymerase. This polymerase can tolerate the high temperature required during PCR and function (optimal activity) in 72^oC, therefore it replaced the DNA polymerase from E. coli which is not able to perform its function in such a high temperature.