Multichange Isothermal Mutagenesis: A New Strategy for Multiple Site-Directed Mutations in Plasmid DNA

Department of Molecular Biology and Genetics and High Throughput Biology Center and ‡Department of Pharmacology, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States.
ACS Synthetic Biology (Impact Factor: 4.98). 03/2013; 2(8). DOI: 10.1021/sb300131w
Source: PubMed

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.

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Available from: Lixin Dai, Dec 15, 2013
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    • "Since 1990, several homologous DNA assembly methods [16-21] have been developed; one method in particular, one-step isothermal in vitro recombination (the ISO method) [20], was used to introduce multiple site-directed mutations [22]. In this method, termed as multichange isothermal in vitro recombination (the MISO method), multiple DNA fragments were first prepared using polymerase chain reaction (PCR) followed by gel purification. "
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