CiteWeb id: 19850000020

CiteWeb score: 9068

DOI: 10.1073/pnas.82.2.488

This chapter focuses on rapid and efficient site-specific mutagenesis without phenotypic selection. The deliberate alteration of DNA sequences by in vitro mutagenesis has become a widely used and invaluable means of probing the structure and function of DNA and the macromolecules for which it codes. After completing the in vitro reactions, uracil can be removed from the template strand by the action of uracil N -glycosylase. To confirm that the colorless mutants contained the expected sequence alteration, DNA from several phage was prepared for sequencing. Incomplete synthesis can result from several factors, including inefficient hybridization of the oligonucleotide primer, inactive (or excess) DNA polymerase, contaminants in the DNA, the polymerase, or the reagents, or a DNA template that contains structures that block polymerization.