Site-specific mutagenesis using synthetic oligodeoxyribonucleotide primers: II. In vitro selection of mutant DNA |
| |
Authors: | Shirley Gillam Michael Smith |
| |
Institution: | Department of Biochemistry, Faculty of Medicine, University of British Columbia, 2075 Wesbrook Place, Vancouver, B.C.Canada V6T 1W5 |
| |
Abstract: | A method for the in vitro selection of mutant DNA has been devised as an adjunct to the recently developed method for the use of short enzymatically-synthesized oligodeoxyribonucleotides of defined sequence as sitespecific mutagens for circular DNA. The selection method uses the mutating oligodeoxyribonucleotide as a primer for Escherichia coli DNA polymerase I (large fragment) under conditions where there is preferential interaction with mutant DNA template. After ligation using T4 DNA ligase, endonuclease Sl is used to degrade single-stranded non-mutant DNA leaving the desired mutant as closed circular duplex DNA. This paper describes the development of the method using mutants in ØX174 DNA as the model system. Studies on the changes A → G and G → A at position 587 of ØX174 viral DNA (am 3 to wild-type and its reversal) show that one or two cycles of selection can lead to a population of phage consisting of close to 100% mutants. |
| |
Keywords: | ØX174 phage large fragment polymerase I S1 endonuclease DNA ligase |
本文献已被 ScienceDirect 等数据库收录! |