Question

At times, a second enzyme is used while the central portion is removed and the stuffer fragment is placed there. Choose the statement which doesn’t holds for this process.

(a) It is a method which is used for avoiding the relegation of central portion before the stuffer fragment is attached over there

(b) In this method, the central portion is cut with another enzyme after being separated from the arms

(c) The arms are also cut with this second enzyme

(d) It is not necessary to remove the fragments generated

This question was posed to me in an internship interview.

My enquiry is from Cloning in Lambda topic in portion Other Vector Systems for E of Genetic Engineering

Answer 1

Correct choice is (c) The arms are also cut with this second enzyme

Best explanation: A second enzyme is used in order to avoid the relegation of the central portion and the arms. In this method, the central portion is cut with a second enzyme after being separated from the arms. These arms are not cut with the second enzymes. Hence the ends created are different now and the arms won’t ligate to the central portion again. It is not necessary to remove the fragments generated because the chances of having a multimolecular reaction which are required for the formation of original phage are very less.