Multiplex-PCR-based recombination as a novel high-fidelity method for directed evolution.

Published

Journal Article

A new and convenient method for the in vitro recombination of single point mutations is presented. This method efficiently reduces the introduction of novel point mutations, which usually occur during recombination processes. A multiplex polymerase chain reaction (multiplex-PCR) generates gene fragments that contain preformed point mutations. These fragments are subsequently assembled into full-length genes by a recombination-PCR step. The process of multiplex-PCR-based recombination (MUPREC) does not require DNase I digestion for gene-fragmentation and is therefore easy to perform, even with small amounts of target DNA. The protocol yields high frequencies of recombination without creating a wild-type background. Furthermore, the low error rate results in high-quality variant libraries of true recombinants, thereby minimizing the screening efforts and saving time and money. The MUPREC method was used in the directed evolution of a Bacillus subtilis lipase that can catalyse the enantioselective hydrolysis of a model meso-compound. Thereby, the method was proved to be useful in producing a reliable second-generation library of true recombinants from which better performing variants were identified by using a high-throughput electrospray ionization mass spectrometry (ESI-MS) screening system.

Full Text

Duke Authors

Cited Authors

  • Eggert, T; Funke, SA; Rao, NM; Acharya, P; Krumm, H; Reetz, MT; Jaeger, K-E

Published Date

  • June 2005

Published In

Volume / Issue

  • 6 / 6

Start / End Page

  • 1062 - 1067

PubMed ID

  • 15880674

Pubmed Central ID

  • 15880674

International Standard Serial Number (ISSN)

  • 1439-4227

Digital Object Identifier (DOI)

  • 10.1002/cbic.200400417

Language

  • eng

Conference Location

  • Germany