Abstract
Site-directed mutagenesis helps unravel the relationship between the structure and function of a protein at the level of amino acid contributions. Many techniques have been established to carry out site-directed mutagenesis on a daily routine. QuikChange II Site-Directed Mutagenesis KitTM is one of such methods available commercially. But this kit is expensive. Here we describe modifications in the transformation step by reducing the usage of competent cells of this protocol that will increase its price-effectiveness. Since the kit is used widely, these modifications might have global application.
Keywords: Site-directed mutagenesis, Transformation, DNA isolation, Competent cells, Chaperone proteins
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 KitTM 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.
Materials and Reagents
Equipment
Procedure
Data analysis
The protocol described above reduces the amount of cells required to perform a transformation using the QuikChange Site-directed mutagenesis kit. The results (Figure 1) show similar cell colonies produced irrespective of the amount of XL1 blue cells used in transformation. The difference in the amount of purified plasmid DNA produced under both conditions was < 20% (Table 1). Therefore the modifications proposed in this protocol are going to increase the price-performance of QuikChange II Site-directed mutagenesis kit. Figure 1. Bacterial colonies produced from transformation of 10 μl (A) or 5 μl (B) XL1 blue cells with the same plasmid at the same concentration. Scale bars = 10 mm. Table 1. The concentration for purified plasmid produced by transforming 10 or 5 μl XL1 blue cells
Recipes
Acknowledgments
We want to thank Golfers Against Cancer, Greensboro, NC for funding (award # A15-0163-001). DFJ is supported by GAC. Contribution: AA developed the idea, interpreted data and wrote the article. DFJ, HK and AA collected data. CPE, MAF and JLS provided reagents. Competing interests: Authors declare no competing conflict of interest.
References
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