Chapter 11, Problem 14P

You sequence a PCR product amplified from a person’s genome, and you see a double peak such as that seen in Fig. 11.11b. Most of the time, this result indicates that the person is a heterozygote for a SNP at that position. But it is also possible that the result is due to a mistake in DNA replication during the PCR amplification, with DNA polymerase misincorporating the wrong nucleotide.

a.	If you saw an artifactual double peak in the sequence trace, did the mistake happen in the first few rounds of PCR amplification or in the last few rounds?
b.	Whether or not you see a double peak, is it more likely that a mistake would happen in the first few rounds of PCR amplification or in the last few rounds?
c.	Given that mistakes can happen during PCR amplification, what could you do to be sure of a person’s genotype? Why would this degree of certainty be difficult to achieve if you were doing preimplantation genotyping of embryos?
d.	PCR relies on heat-stable DNA polymerases from thermophilic bacteria that grow in hot springs. The DNA polymerase originally used for PCR, from the bacterium Thermus aquaticus, lacks the 3′-to-5′ exonuclease found in other DNA polymerases such as that from E. coli (review Fig. 7.9). Why do scientists now most often use DNA polymerase from a different thermophilic bacterium (Pyrococcus furiosa) that does contain this exonuclease function?