Isopentenyl diphosphate isomerase. Site-directed mutagenesis of Cys139 using "counter" PCR amplification of an expression plasmid

Abstract

Site-directed mutagenesis of Cys139 in yeast isopentenyl diphosphate isomerase was accomplished by "counter" polymerase chain reaction amplification of the gene in expression plasmid plPS241 using only two DNA primers. One primer complemented a region of the sense strand and contained the mutations. The second primer was an exact complement of the antisense strand. When hybridized to the plasmid template, the 5′-ends of the primers butted against one another, and PCR amplification resulted in synthesis of double strand linear mutant expression plasmid. The linear DNA was circularized by blunt end ligation to yield an expression plasmid that overproduced mutant yeast enzyme in transformed E. ..