Release Time:2019-03-10  Hits:

Indexed by: Journal Article

Date of Publication: 2008-07-01

Journal: BIOTECHNOLOGY LETTERS

Included Journals: EI、SCIE

Volume: 30

Issue: 7

Page Number: 1227-1232

ISSN: 0141-5492

Key Words: AdoMet synthetase; directed evolution; DNA recombination; error-prone PCR; S-adenosylmethionine

Abstract: An efficient method for creating a DNA library is presented in which gene mutagenesis and recombination can be introduced by integrating error-prone PCR with a staggered extension process in one test tube. In this process, less than 15 cycles of error-prone PCR are used to introduce random mutations. After precipitated and washed with ethanol solution, the error-prone PCR product is directly used both as template and primers in the following staggered extension process to introduce DNA recombination. The method was validated by using adenosyl-methionine (AdoMet) synthetase gene, sam1, as a model. The full-length target DNA fragment was available after a single round. After being selected with a competitive inhibitor, ethionine, a mutated gene was obtained that increased AdoMet accumulation in vivo by 56%.