Question

Which of the following is not a step involved in site directed mutagenesis using a hypothetical example?

(a) Derive the primary structure of the enzyme from the DNA sequence

(b) Identification of mutant DNA by hybridization with radioactively labelled oligonucleotides

(c) Cleavage by Hind III

(d) Primary structure of putative enzyme is proposed with an asparagine residue replacing the serine present in the native enzyme

This question was addressed to me in exam.

My enquiry is from Future Prospects topic in section Enzyme Technology Future Prospects of Enzyme Technology

Answer 1

Correct answer is (c) Cleavage by Hind III

Easiest explanation: Cleavage by Hind III is a step involved in production of penicillin – G – amidases and not site directed mutagenesis. The steps involved in site directed mutagenesis are as follows:

• Derive the primary structure of the enzyme from the DNA sequence.

• Primary structure of putative enzyme is proposed with an asparagine residue replacing the serine present in the native enzyme.

• Synthesis of primer which is complementary to a section of the gene apart from the base mismatch.

• The oligonucleotide primer is annealed to a single-stranded copy of the gene and polymerase chain reaction occurs which produces both mutant and wild-type clones.

• Identification of mutant DNA by hybridization with radioactively labelled oligonucleotides.