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

Leslie A. Mitchell, Yizhi Cai, Martin Taylor, Anne Marie Noronha, James Chuang, Lixin Dai, Jef D. Boeke

    Research output: Contribution to journalArticlepeer-review

    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.

    Original languageEnglish (US)
    Pages (from-to)473-477
    Number of pages5
    JournalACS Synthetic Biology
    Volume2
    Issue number8
    DOIs
    StatePublished - Aug 16 2013

    Keywords

    • QuikChange
    • deletion
    • insertion
    • one-step isothermal assembly
    • point mutation
    • site-directed mutagenesis

    ASJC Scopus subject areas

    • Biomedical Engineering
    • Biochemistry, Genetics and Molecular Biology (miscellaneous)

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