[6] Efficient site-directed mutagenesis using uracil-containing DNA

Oligonucleotide-directed mutagenesis is a widely used procedure for studying the structure and function of DNA and the macromolecules for which it codes. The most commonly used strategy for site-directed mutagenesis is to clone the segment of DNA to be mutated into a vector whose DNA can be obtained in single-stranded form. An oligonucleotide partially complementary to the region to be altered, but containing the mutation to be introduced, is hybridized to the single-stranded DNA. A complementary strand is synthesized by DNA polymerase using the oligonucleotide as a primer. The efficiency of site-directed mutagenesis, that is, the proportion of progeny containing the desired sequence alteration, depends on the quality of each of the steps in the procedure. The number of progeny clones that must be monitored to obtain the desired mutant increases as the efficiency of mutagenesis decreases.