A gradient PCR-based screen for use in site-directed mutagenesis.
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.
Padmakumar
V C
VC
Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India.
Varadarajan
Raghavan
R
D017931:DNA Primers / Q000235:genetics; D005784:Gene Amplification / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D013696:Temperature; D013698:Templates, Genetic
D017931:DNA Primers