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ACS Synth Biol. 2013 Aug 16;2(8):473-7. doi: 10.1021/sb300131w. Epub 2013 Mar 11.

Multichange isothermal mutagenesis: a new strategy for multiple site-directed mutations in plasmid DNA.

Author information

1
Department of Molecular Biology and Genetics and High Throughput Biology Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA. lmitch24@jhmi.edu.

Abstract

Multichange ISOthermal (MISO) mutagenesis is a new technique allowing simultaneous introduction of multiple site-directed mutations into plasmid DNA by leveraging two existing ideas: QuikChange-style primers and one-step isothermal (ISO) assembly. Inversely partnering pairs of QuikChange primers results in robust, exponential amplification of linear fragments of DNA encoding mutagenic yet homologous ends. These products are amenable to ISO assembly, which efficiently assembles them into a circular, mutagenized plasmid. Because the technique relies on ISO assembly, MISO mutagenesis is additionally amenable to other relevant DNA modifications such as insertions and deletions. Here we provide a detailed description of the MISO mutagenesis concept and highlight its versatility by applying it to three experiments currently intractable with standard site-directed mutagenesis approaches. MISO mutagenesis has the potential to become widely used for site-directed mutagenesis.

PMID:
23654272
PMCID:
PMC4040258
DOI:
10.1021/sb300131w
[Indexed for MEDLINE]
Free PMC Article

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