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A rapid, efficient, and economical inverse polymerase chain reaction-based method for generating a site saturation mutant library

Jain, Pankaj C and Varadarajan, Raghavan (2014) A rapid, efficient, and economical inverse polymerase chain reaction-based method for generating a site saturation mutant library. In: ANALYTICAL BIOCHEMISTRY, 449 . pp. 90-98.

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Official URL: http://dx.doi.org/10.1016/j.ab.2013.12.002

Abstract

With the development of deep sequencing methodologies, it has become important to construct site saturation mutant (SSM) libraries in which every nucleotide/codon in a gene is individually randomized. We describe methodologies for the rapid, efficient, and economical construction of such libraries using inverse polymerase chain reaction (PCR). We show that if the degenerate codon is in the middle of the mutagenic primer, there is an inherent PCR bias due to the thermodynamic mismatch penalty, which decreases the proportion of unique mutants. Introducing a nucleotide bias in the primer can alleviate the problem. Alternatively, if the degenerate codon is placed at the 5' end, there is no PCR bias, which results in a higher proportion of unique mutants. This also facilitates detection of deletion mutants resulting from errors during primer synthesis. This method can be used to rapidly generate SSM libraries for any gene or nucleotide sequence, which can subsequently be screened and analyzed by deep sequencing. (C) 2013 Elsevier Inc. All rights reserved.

Item Type: Journal Article
Additional Information: Copyright for this article belongs to the ACADEMIC PRESS INC ELSEVIER SCIENCE, USA
Keywords: Site-directed mutagenesis; PCR bias; Primer design; High throughput; Base mismatch
Department/Centre: Division of Biological Sciences > Molecular Biophysics Unit
Depositing User: Id for Latest eprints
Date Deposited: 21 May 2014 07:46
Last Modified: 21 May 2014 07:46
URI: http://eprints.iisc.ac.in/id/eprint/48943

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