title: A rapid, efficient, and economical inverse polymerase chain reaction-based method for generating a site saturation mutant library.
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.
authors: Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India.
authors: Pankaj C
authors: PC
authors: Jain
authors: Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India; Jawaharlal Nehru Center for Advanced Scientific Research, Bangalore 560 004, India. Electronic address: varadar@mbu.iisc.ernet.in.
authors: Raghavan
authors: R
authors: Varadarajan
mesh_terms: D020137:Base Pair Mismatch; D001483:Base Sequence; D003062:Codon; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction / Q000191:economics / Q000379:methods*
chemical_list: D003062:Codon; D017931:DNA Primers
keywords: Base mismatch; High throughput; PCR bias; Primer design; Site-directed mutagenesis