A gradient PCR-based screen for use in site-directed mutagenesis

• Published in: Analytical biochemistry 2003-03, Vol.314 (2), p.310-315
• Main Authors: Padmakumar, V.C, Varadarajan, Raghavan
• Format: Article
• Language: English
• Subjects: DNA Primers - genetics; Gene Amplification - genetics; Mutagenesis, Site-Directed; Mutation; Plasmids - genetics; Polymerase Chain Reaction - methods; Temperature; Templates, Genetic
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/S0003-2697(02)00688-7

Site-directed mutagenesis is widely used to study protein and nucleic acid structure and function. Despite recent advancements in the efficiency of procedures for site-directed mutagenesis, the fraction of site-directed mutants by most procedures rarely exceeds 50% on a routine basis and is never 100%. Hence it is typically necessary to sequence two or three clones each time a site-directed mutant is constructed. We describe a simple and robust gradient-PCR-based screen for distinguishing site-directed mutants from the starting, unmutated plasmid. The procedure can use either purified plasmid DNA or colony PCR, starting from a single colony. The screen utilizes the primer used for mutagenesis and a common outside primer that can be used for all other mutants constructed with the same template. Over 30 site-specific mutants in a variety of templates were successfully screened and all of the mutations detected were subsequently confirmed by DNA sequencing. A single base pair mismatch could be detected in an oligonucleotide of 36 bases. Detection efficiency was relatively independent of starting template concentration and the nature of the outside primer used.