Abstract
BackgroundIn protein engineering, site-directed mutagenesis methods are used to generate DNA sequences with mutated codons, insertions or deletions. In a widely used method, mutations are generated by PCR using a pair of oligonucleotide primers designed with mismatching nucleotides at the center of the primers. In this method, primer-primer annealing may prevent cloning of mutant cDNAs. To circumvent this problem we developed an alternative procedure that does not use forward-reverse primer pair in the same reaction.ResultsIn initial studies we used a double-primer PCR mutagenesis protocol, but sequencing of products showed tandem repeats of primer in cloned DNA. We developed an alternative method that starts with two Single-Primer Reactions IN Parallel using high-fidelity Pwo DNA polymerase. Thus, we call the method with the acronym SPRINP. The SPRINP reactions are then combined, denatured at 95°C, and slowly cooled, promoting random annealing of the parental DNA and the newly synthesized strands. The products are digested with DpnI that digests methylated parental strands, and then transformed into E. coli. Using this method we generated >40 mutants in cDNAs coding for human Epithelial Na+ Channel (ENaC) subunits. The method has been tested for 1–3 bp codon mutation and insertion of a 27 bp epitope tag into cDNAs.ConclusionThe SPRINP mutagenesis protocol yields mutants reliably and with high fidelity. The use of a single primer in each amplification reaction increases the probability of success of primers relative to previous methods employing a forward and reverse primer pair in the same reaction.
Highlights
In protein engineering, site-directed mutagenesis methods are used to generate DNA sequences with mutated codons, insertions or deletions
We developed a Site-directed mutagenesis (SDM) method that includes two PCR reactions run in parallel with each one of the forward and reverse primers
SDM using double-primer PCR In initial studies we tried to generate mutants following a widely used double-primer PCR procedure outlined in Methods
Summary
Site-directed mutagenesis methods are used to generate DNA sequences with mutated codons, insertions or deletions. In a widely used method, mutations are generated by PCR using a pair of oligonucleotide primers designed with mismatching nucleotides at the center of the primers. In this method, primer-primer annealing may prevent cloning of mutant cDNAs. In this method, primer-primer annealing may prevent cloning of mutant cDNAs To circumvent this problem we developed an alternative procedure that does not use forward-reverse primer pair in the same reaction. Site-directed mutagenesis (SDM) methods are used to generate cloned DNAs with modified sequences for examining the importance of specific residues in protein structure and function. The use of complementary primer pairs may lead (page number not for citation purposes)
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