Question

In site-directed mutagenesis, how is the single base change introduced into the cloned gene sequence? Please select the most correct answer.

	A.	Two of these can be used.
	B.	All of these will work for site-directed mutagenesis.
	C.	The single base is changed by using PCR and a PCR primer which is complementary to the region of the gene to be changed. The primer is altered by a single base, and the PCR product of the gene and the plasmid will then carry that single base change in the gene.
	D.	The single base is changed by using PCR and dideoxynucleotide for that base, and the PCR product of the gene and the plasmid will then carry that single base change in the gene.
	E.	The single base is changed by using CRISPR/CAS to target a specific DNA sequence for substitution mutations which will substitute the wildtype base with the new base.

Answer 1

The most correct answer is A, two are correct. The correct choices are C and E.  

Site-directed mutagenesis involves changes at a single location/basepair of the target sequence and can be achieved by PCR methods which normally use primers ( one or two primers with the changed sequence can be used) which have the required change incorporated.

Another method uses CRISPR/Cas9 in a PCR free method with the help of single-guide RNAs.

However, the use of dideoxynucleotides inhibits the chain elongation in PCR and is typically used in sequencing experiments using Sangers method and cannot be used for site-directed mutagenesis.