REPLACR-mutagenesis, a one-step method for site-directed mutagenesis by recombineering
Mutagenesis is an important tool to study gene regulation, model disease-causing mutations and for functional characterisation of proteins. Most of the current methods for mutagenesis involve multiple step procedures. One of the most accurate methods for genetically altering DNA is recombineering ,...
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Veröffentlicht in: | Scientific reports 2016-01, Vol.6 (1), p.19121, Article 19121 |
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Format: | Artikel |
Sprache: | eng |
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Zusammenfassung: | Mutagenesis is an important tool to study gene regulation, model disease-causing mutations and for functional characterisation of proteins. Most of the current methods for mutagenesis involve multiple step procedures. One of the most accurate methods for genetically altering DNA is
recombineering
, which uses bacteria expressing viral recombination proteins. Recently, the use of
in vitro
seamless assembly systems using purified enzymes for multiple-fragment cloning as well as mutagenesis is gaining ground. Although these
in vitro
isothermal reactions are useful when cloning multiple fragments, for site-directed mutagenesis it is unnecessary. Moreover, the use of purified enzymes
in vitro
is not only expensive but also more inaccurate than the high-fidelity recombination inside bacteria. Here we present a single-step method, named REPLACR-mutagenesis (
R
ecombineering of
E
nds of linearised
PLA
smids after P
CR
), for creating mutations (deletions, substitutions and additions) in plasmids by
in vivo
recombineering. REPLACR-mutagenesis only involves transformation of PCR products in bacteria expressing Red/ET recombineering proteins. Modifications in a variety of plasmids up to bacterial artificial chromosomes (BACs; 144 kb deletion) have been achieved by this method. The presented method is more robust, involves fewer steps and is cost-efficient. |
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ISSN: | 2045-2322 2045-2322 |
DOI: | 10.1038/srep19121 |