The polymerase chain reaction (PCR) is a powerful biological tool that allows the rapid amplification of any fragment of DNA without purification. In PCR, DNA primers are made to flank the specific DNA sequence to be amplified. These primers are then extended to the end of the DNA molecule with the use of a heat-resistant DNA polymerase. The newly synthesized DNA strand is then used as the template to undergo another round of replication.
The 1st step in PCR is the melting of the target DNA into 2 single strands by heating the reaction mixture to approximately 94° C, and then rapidly cooling the mixture to allow annealing of the DNA primers to their specific locations. Once the primer has annealed, the temperature is elevated to 72° C to allow optimal activity of the DNA polymerase. The polymerase will continue to add nucleotides until the entire complimentary strand of the template is completed at which point the cycle is repeated (Figure 1)
Figure 1
One of the uses of PCR is sex determination, which requires amplification of intron 1 of the amelogenin gene. This gene found on the X-Y homologous chromosomes has a 184 base pair deletion on the Y homologue. Therefore, by amplifying intron 1 females can be distinguished from males by the fact that males will have 2 different sizes of the amplified DNA while females will only have 1 unique fragment size.
Why is a heat resistant DNA polymerase required for successive replication in the polymerase chain reaction, rather than simply a human DNA polymerase?
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Reference / correct answer:
The high temperatures required to melt the DNA double strand may denature a normal human cellular DNA polymerase.
Since mammalian cells function at 37° C, this is also the optimal temperature for enzyme activity. The temperatures used in PCR, which are well above 70° C, would easily denature human DNA polymerase which is why a heat resistant DNA polymerase is required. Using human DNA polymerase would require new enzyme after each cycle, and therefore would not be very efficient.
