Open reading frame analysis by selective PCR-mediated deletion mutagenesis

Abstract

Recently, we demonstrated that a nested set of DNA fragments can be obtained by using one specific primer and one semirandom primer in a polymerase chain reaction (PCR). We now describe a strategy for selective deletion mutagenesis that is based on this observation. The gene of interest is cloned as a fusion construct with a selectable marker in a small vector, allowing for PCR amplification of the entire recombinant plasmid. The specific primer is complementary to the vector sequence beyond the gene of interest and is oriented downstream. The 3′ end of the semirandom primer is complementary to a triplet (GAT) that is scattered over the entire open reading frame (ORF). It is shown by nucleotide sequence analysis that deletion mutants result exclusively from annealing of the semirandom primer at different GAT triplets. PCR products resulting fro from annealing to GAT triplets elsewhere in the plasmid are counterselected by the need for replication functions and for the expression of the selectable marker. This technique is demonstrated on the Saccharomyces cerevisiae ORF YCL56C.