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- Title
A rapid and efficient method for multiple-site mutagenesis with a modified overlap extension PCR.
- Authors
Yingfeng An; Jianfei Ji; Wenfang Wu; Anguo Lv; Ribo Huang; Yutuo Wei
- Abstract
A rapid and efficient method to perform site-directed mutagenesis based on an improved version of overlap extension by polymerase chain reaction (OE-PCR) is demonstrated in this paper. For this method, which we name modified (M)OE-PCR, there are five steps: (1) synthesis of individual DNA fragments of interest (with average 20-bp overlap between adjacent fragments) by PCR with high-fidelity pfu DNA polymerase, (2) double-mixing (every two adjacent fragments are mixed to implement OE-PCR without primers), (3) pre-extension (the teams above are mixed to obtain full-length reassembled DNA by OE-PCR without primers), (4) synthesis of the entire DNA of interest by PCR with outermost primers and template DNA from step 3, (5) post-extension (ten cycles of PCR at 72°C for annealing and extension are implemented). The method is rapid, simple and error-free. It provides an efficient choice, especially for multiple-site mutagenesis of DNAs; and it can theoretically be applied to the modification of any DNA fragment. Using the MOE-PCR method, we have successfully obtained a modified sam1 gene with eight rare codons optimized simultaneously.
- Subjects
DNA; GENES; NUCLEIC acids; MUTAGENESIS; GENETIC mutation; RADIOGENETICS; DNA polymerases; POLYMERASE chain reaction
- Publication
Applied Microbiology & Biotechnology, 2005, Vol 68, Issue 6, p774
- ISSN
0175-7598
- Publication type
Article
- DOI
10.1007/s00253-005-1948-8