Journal
ACS SYNTHETIC BIOLOGY
Volume 2, Issue 8, Pages 473-477Publisher
AMER CHEMICAL SOC
DOI: 10.1021/sb300131w
Keywords
site-directed mutagenesis; one-step isothermal assembly; QuikChange; point mutation; insertion; deletion
Categories
Funding
- National Science Foundation [MCB-1026068]
- DARPA [N66001-12-C-4020]
- Div Of Molecular and Cellular Bioscience
- Direct For Biological Sciences [1443299, 1441866] Funding Source: National Science Foundation
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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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