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 journalArticle

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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  • Cite this

    Mitchell, L. A., Cai, Y., Taylor, M., Noronha, A. M., Chuang, J., Dai, L., & Boeke, J. D. (2013). Multichange isothermal mutagenesis: A new strategy for multiple site-directed mutations in plasmid DNA. ACS Synthetic Biology, 2(8), 473-477. https://doi.org/10.1021/sb300131w