Answer to Question #263444 in Molecular Biology for Eves

Question

Why is the generation of single and double mutants were done using PCR-based QuikChnage site-directed mutagenesis?

Answer 1

Mutagenesis is commonly used to understand the relationship between gene regulatory regions and protein structure and function. Depending on the number of sites to mutate, site-directed mutagenesis can be divided into two types: single or multiple mutations. For single mutations, this method is based on amplification of double-stranded DNA from a plasmid using complementary oligonucleotides with the mutation of interest. Due to its simplicity, low time spent, and high efficiency, this is one of the most common strategies for introducing mutations into DNA fragments. For multiple mutations, the method either integrates the desired mutation into the same reaction at the same time or is obtained after several mutations.

 Many commercial kits are available for simple mutagenesis. These kits are easy to use, but struggle to get large deletions on a regular basis.