Modifications in the Transformation Step of Commercially Available Site-directed Mutagenesis Kit Increase Its Price-performance

Background

Physiological function of a protein is meticulously controlled by its structure. Conformation changes in a protein from its native state have disastrous implications on human health (Carrell and Gooptu, 1998; Chiti and Dobson, 2006). For example, a single amino acid change in hemoglobin leads to sickle cell disease (Ingram, 2004) and a single amino acid change in -synuclein is the reason for familial parkinsonism (Krüger et al., 1998). Thus, study of amino acids in relation to protein structure and function is highly relevant and useful with respect to human health.

In 1982 Winter et al., devised a method for selective replacement of single amino acid in a protein, tyrosyl tRNA synthetase. In their site-directed replacement, they mutated Cys35 of the molecule to Ser and observed that single amino acid change caused reduction in the tyrosyl tRNA synthetase enzyme activity (Winter et al., 1982). Since then many different methods have been developed for achieving site-directed mutagenesis of proteins. QuikChange mutagenesis method is one such method (Papworth et al., 1994; Braman et al., 1996). Further, in using QuikChange II Site-Directed Mutagenesis Kit^TM from Agilent Technologies, the need for specific restriction sites and the strand status is completely eliminated and the process can be completed in a short period of time. However, QuikChange II Site-Directed Mutagenesis Kit is expensive and cost to product ratio is very high. In this study, we describe modifications in the transformation step of the protocol, which will significantly reduce the use of materials in carrying out each site-directed mutation and will also reduce the time taken in the whole process. These modifications will maximize the yield of QuikChange II Site-Directed Mutagenesis Kit and will significantly improve its price-performance ratio.