Rapid and reliable site directed mutagenesis using Kunkel's approach.
Oligonucleotide based site directed mutagenesis (SDM) is an invaluable technique in molecular biology. Among the various methods developed for SDM, the PCR-based approach, Kunkel's and the Eckstein's procedures are widely used. The Kunkel's method, on account of its cost effectiveness and simplicity, is preferred by many a scientist. However, a general drawback of this method is the high background due to persistence of the parent template resulting in low efficiency of mutagenesis. In this report, we describe a modification of the Kunkel's method to increase the efficiency of selecting against the wild type strand. We have used Sequenase for the extension reaction, and introduced an in vitro UDG step to enhance the biological selection against the parent strand. Consequently, the efficiency of the modified method is enhanced to allow screening of the mutants directly by DNA sequencing. A step by step single tube protocol which is over in less than three hours makes it a method of choice for efficient and cost-effective site directed mutagenesis.
Handa
P
P
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India.
Varshney
U
U
D004259:DNA-Directed DNA Polymerase; D004926:Escherichia coli; D005821:Genetic Techniques; D007202:Indicators and Reagents; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction / Q000379:methods; D015203:Reproducibility of Results; D013698:Templates, Genetic
D007202:Indicators and Reagents; C034970:bacteriophage T7 induced DNA polymerase; D004259:DNA-Directed DNA Polymerase