ArticleScientific reports2016
REPLACR-mutagenesis, a one-step method for site-directed mutagenesis by recombineering.
Article in Scientific reports, 2016. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 17 papers.
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Who cites it
17 citing papers in PubMed, 51 citations in OpenAlex.
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- Influence of expression and purification protocols on Gα biochemical activity: kinetics of plant and mammalian G protein cycles.Frontiers in molecular biosciences · 2025Article
- The rise and future of CRISPR-based approaches for high-throughput genomics.FEMS microbiology reviews · 2024Review
- A simplified Gibson assembly method for site directed mutagenesis by re-use of standard, and entirely complementary, mutagenesis primers.BMC biotechnology · 2022Article
- A high-efficiency method for site-directed mutagenesis of large plasmids based on large DNA fragment amplification and recombinational ligation.Scientific reports · 2021Article
- An Improved Vector System for Homogeneous and Stable Gene Regulation.International journal of molecular sciences · 2021Article
- A two-step PCR assembly for construction of gene variants across large mutational distances.Biology methods & protocols · 2021Article
- Genetically Engineered Lung Cancer Cells for Analyzing Epithelial-Mesenchymal Transition.Cells · 2019Article
- CRISPR/Cas9-mediated gene deletion of the ompA gene in symbiotic Cedecea neteri impairs biofilm formation and reduces gut colonization of Aedes aegypti mosquitoes.PLoS neglected tropical diseases · 2019Article
- The Pathogenic TSH β-subunit Variant C105Vfs114X Causes a Modified Signaling Profile at TSHR.International journal of molecular sciences · 2019Article
- Review
- Efficient strategy for introducing large and multiple changes in plasmid DNA.Scientific reports · 2018Article
- A restriction-free method for gene reconstitution using two single-primer PCRs in parallel to generate compatible cohesive ends.BMC biotechnology · 2017Article
- Article
- IVA cloning: A single-tube universal cloning system exploiting bacterial In Vivo Assembly.Scientific reports · 2016Article
Corrections and comments
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Authors and funding
6 authors at 2 institutions in 3 countries.
Funding
No grant is acknowledged in the PubMed record.
Abstract
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 (Recombineering of Ends of linearised PLAsmids after PCR), 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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