title: Rapid and efficient site-specific mutagenesis without phenotypic selection.
abstract: Several single-base substitution mutations have been introduced into the lacZ alpha gene in cloning vector M13mp2, at 40-60% efficiency, in a rapid procedure requiring only transfection of the unfractionated products of standard in vitro mutagenesis reactions. Two simple additional treatments of the DNA, before transfection, produce a site-specific mutation frequency approaching 100%. The approach is applicable to phenotypically silent mutations in addition to those that can be selected. The high efficiency, approximately equal to 10-fold greater than that observed using current methods without enrichment procedures, is obtained by using a DNA template containing several uracil residues in place of thymine. This template has normal coding potential for the in vitro reactions typical of site-directed mutagenesis protocols but is not biologically active upon transfection into a wild-type (i.e., ung+) Escherichia coli host cell. Expression of the desired change, present in the newly synthesized non-uracil-containing covalently closed circular complementary strand, is thus strongly favored. The procedure has been applied to mutations introduced via both oligonucleotides and error-prone polymerization. In addition to its utility in changing DNA sequences, this approach can potentially be used to examine the biological consequences of specific lesions placed at defined positions within a gene.
mesh_terms: D001483:Base Sequence; D003090:Coliphages / Q000235:genetics; D045647:DNA Glycosylases; D004277:DNA, Single-Stranded / Q000032:analysis; D004279:DNA, Viral / Q000032:analysis*; D004926:Escherichia coli / Q000235:genetics; D009154:Mutation; D009699:N-Glycosyl Hydrolases / Q000378:metabolism; D010641:Phenotype; D014162:Transfection; D014498:Uracil / Q000378:metabolism; D051981:Uracil-DNA Glycosidase
chemical_list: D004277:DNA, Single-Stranded; D004279:DNA, Viral; D014498:Uracil; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase