Question

During amplification, there are chances of having a product of a mixture of different sequences. There are various ways to detect it. Which of the statement is true in regard to it?

(a) Direct sequencing can’t be used in the case if the template DNA is heterozygous at the locus

(b) Direct sequencing can be used if the template DNA is heterozygous at the locus

(c) If cloning is done before sequencing, then it is detected via using only a single clone for sequencing

(d) In the case several recombinants are used, it can’t go undetected

I have been asked this question by my college director while I was bunking the class.

My enquiry is from Precautions and Drawbacks in division Polymerase Chain Reaction of Genetic Engineering

Answer 1

The correct option is (b) Direct sequencing can be used if the template DNA is heterozygous at the locus

Best explanation: If the template DNA is heterozygous at the locus, it can be detected via using direct sequencing. It is so because it would give rise to two different signals at the same nucleotide position at the sequence output. If the cloning is done before sequencing, a single clone won’t be helpful to detect heterogeneity. It is because single clones are derived from a single PCR product. Also, if several recombinants are used, there are chances that they go undetected.