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• W2807350372 abstract "Abstract While the QuikChange site-directed mutagenesis method and its later modifications are extremely useful and simple, they suffer from several drawbacks. Here, we propose a new method, named LFEAP mutagenesis ( L igation of F ragment E nds A fter P CR) for creating various mutations in plasmid by leveraging three existing concepts: inverse PCR, single primer PCR, and sticky-end assembly. The first inverse PCR on the target plasmid yielded linearized DNA fragments with mutagenic ends, and a second single primer PCR resulted in complementary single-stranded DNA fragments with the addition of overhangs at the 5′ end of each strand. The resulting single strands were then annealed to produce double-stranded DNA with free 5′ single-stranded DNA tails. These products with compatible sticky ends were efficiently assembled into a circular, mutagenized plasmid. With this strategy, multiple simultaneous changes (up to 15) and mutations in large plasmids (up to 50 kb) were achieved with high efficiency and fidelity. LFEAP mutagenesis is a versatile method that offers significant advantages for introducing large and multiple changes in plasmid DNA." @default.
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• W2807350372 date "2018-01-29" @default.
• W2807350372 modified "2023-10-15" @default.
• W2807350372 title "Efficient strategy for introducing large and multiple changes in plasmid DNA" @default.
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• W2807350372 doi "https://doi.org/10.1038/s41598-018-20169-8" @default.
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