A General Method for Saturation Mutagenesis of Cloned DNA Fragments
1985; American Association for the Advancement of Science; Volume: 229; Issue: 4710 Linguagem: Inglês
10.1126/science.2990046
ISSN1095-9203
AutoresR Myers, Leonard S. Lerman, Tom Maniatis,
Tópico(s)Bacterial Genetics and Biotechnology
ResumoA new procedure for generating and isolating random single-base substitutions in cloned DNA fragments is presented. The mutations are generated by treatment of single-stranded DNA with various chemicals, followed by the synthesis of the complementary strand with reverse transcriptase. Misincorporation frequently occurs when the enzyme encounters a damaged base in the mutagenized template DNA. The resulting duplex DNA fragments containing random single-base substitutions are cloned, amplified as a population, and isolated from wild-type DNA by preparative denaturing gradient gel electrophoresis. The physical separation of mutant DNA fragments makes it possible to isolate and characterize large numbers of site-directed single-base substitutions in the absence of a phenotypic selection. This procedure should be generally applicable to the fine-structure genetic analysis of regulatory and protein-coding sequences.
Referência(s)