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Rapid and reliable site directed mutagenesis using Kunkel's approach

Handa, P and Varshney, U (1998) Rapid and reliable site directed mutagenesis using Kunkel's approach. In: Indian Journal of Biochemistry and Biophysics, 35 (02). pp. 63-66.

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Official URL: http://www.ncbi.nlm.nih.gov/pubmed/9753863?ordinal...

Abstract

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.

Item Type: Journal Article
Additional Information: Copyright of this article belongs to National Institute of Science Communication and Information Resources.
Department/Centre: Division of Biological Sciences > Microbiology & Cell Biology
Date Deposited: 03 Dec 2009 11:23
Last Modified: 03 Dec 2009 11:23
URI: http://eprints.iisc.ernet.in/id/eprint/19109

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