Question

Which one of the following is not done if amplification of the non flanking region is carried out?

(a) Firstly, restriction enzyme digestion is done of those sites whose sequence is not known

(b) Then the self-ligation of molecules is allowed

(c) Now the molecules are cleaved where the known sequence is

(d) Again, the molecules are linearized and the known sequence is in the middle

This question was addressed to me at a job interview.

I'd like to ask this question from Modifications in PCR topic in section Polymerase Chain Reaction of Genetic Engineering

Answer 1

The correct option is (d) Again, the molecules are linearized and the known sequence is in the middle

To explain I would say: If such amplification has to be carried out, firstly the restriction enzyme digestion is done at those sites whose sequence is not known till now. Then self ligation is carried out i.e. conditions for intramolecular ligation is applied. As the molecules circularize now, cleavage is carried out of known sequence. As the known sequence is cleaved, the molecule is now cleaved with a known sequence at the ends. As it known sequence, primers can be constructed for it and the unknown region is amplified.