A METHOD FOR THE SITE-DIRECTED MONO-MUTAGENESIS AND MULTI-MUTAGENESISOF DOUBLE-STRANDED DNA

A general solid-phase method for the site-directed mutagenesis of doub le-stranded DNA (dsDNA) is described. Plasmid DNA is linearized using either a restriction endonuclease (ENase) or the RecA-assisted ENase o r RecA-AC cleavage method. Alternatively, PCR may be used to generate linear dsDNA. One or both strands of the DNA is biotinylated and attac hed to a solid support, and the DNA strands are separated using 0.2 M NaOH. An extension oligodeoxyribonucleotide (oligo) and a single or mu ltiple oligo(s) containing the desired mutation(s) are annealed to one of the bound DNA strands and used to initiate the synthesis of a comp lementary strand by a nonstrand-displacing DNA polymerase. The in vitr o synthesized strand incorporating the desired alteration(s) is melted off of the support and recircularized using one of several types of b ridging oligos, DNA ligase, and a DNA polymerase and transformed into the host. Greater than 90% mutagenic efficiency has been obtained usin g this method.