DNA gap repair in Escherichia coli for multiplex site-directed mutagenesis

George T. Lyozin, Luca Brunelli

Research output: Contribution to journalArticle

Abstract

Site-directed mutagenesis allows the generation of novel DNA sequences that can be used for a variety of important applications such as the functional analysis of genetic variants. To overcome the limitations of existing site-directed mutagenesis approaches, we explored in vivo DNA gap repair. We found that site-specific mutations in plasmid DNA can be generated in Escherichia coli using mutant single-stranded oligonucleotides to target PCR-derived linear double-stranded plasmid DNA. We called this method DeGeRing, and we characterized its advantages, including non-biased multiplex mutagenesis, over existing site-directed mutagenesis methods such as recombineering (recombination-mediated genetic engineering), single DNA break repair (SDBR, introduced by W. Mandecki), and QuikChange (Agilent Technologies, La Jolla, CA). We determined the efficiency of DeGeRing to induce site-directed mutations with and without a phenotype in three K-12 E coli strains using multiple single-stranded oligonucleotides containing homological and heterological parts of various sizes. Virtual lack of background made the isolation of mutants with frequencies up to 10-6 unnecessary. Our data show that endogenous DNA gap repair in E coli supports efficient multiplex site-directed mutagenesis. DeGeRing might facilitate the generation of mutant DNA sequences for protein engineering and the functional analysis of genetic variants in reverse genetics.

Original languageEnglish (US)
Pages (from-to)6351-6368
Number of pages18
JournalFASEB Journal
Volume34
Issue number5
DOIs
StatePublished - May 1 2020

Keywords

  • gap repair cloning
  • in vivo DNA engineering
  • protein engineering
  • reverse genetics

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Genetics

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