Site-specific mutagenesis by error-directed DNA synthesis

Abstract

An important experimental strategy for defining functional regions of a DNA sequence involves deleting or mutating particular sequences in vitro and observing the effects of the changes in a functional assay. We have approached the problem of making specific changes at defined sites in a nucleotide sequence by attempting to make use of the mistakes made by DNA polymerase enzymes while copying a sequence. Our approach is to initiate DNA synthesis from an isolated restriction fragment, and to elongate the fragment to a designated position by sequentially adding complementary nucleotides, then to incorporate the non-complementary nucleotide using an error-prone DNA polymerase followed by continuation of faithful synthesis. Here we demonstrate by DNA sequence analysis that both transitions and transversions can be produced at a designated position on the DNA template using this method.