You sequence a PCR product amplified from a person\'s genome, and you see a doub

Question

You sequence a PCR product amplified from a person's genome, and you see a double peak such as that seen in Fig. 10.11b on p. 350. 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 a 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 ever 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?

Explanation / Answer

a. The mistake must have happened during the early PCR cycles. If the mistake happens during late PCR cycles, it would not produce a clear peak as the number of products would be less.

b. Irrespective of the double peak presence, mistakes that occur only during early cycles of PCR would be visualized in the sequencing diagram.

c. After sequencing, we can perform restriction enzyme digestion specific to the SNP to confirm its presence. This is a difficult task because we can not isolate PCR fragments and set up digestion for all of them at the whole genome level (The number of SNPs is generally in the range of thousands).

Related Questions

Navigate

• Browse (All)
• Browse Y
• Subjects

---

---

Integrity-first tutoring: explanations and feedback only — we do not complete graded work. Learn more.