A rapid and efficient method for multiple-site mutagenesis with a modified overlap extension PCR.
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 degrees 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.
An
Yingfeng
Y
Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang, China.
Ji
Jianfei
J
Wu
Wenfang
W
Lv
Anguo
A
Huang
Ribo
R
Wei
Yutuo
Y
D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D013698:Templates, Genetic; D013997:Time Factors
D017931:DNA Primers; D004269:DNA, Bacterial; D004271:DNA, Fungal; C072146:Pfu DNA polymerase; D004259:DNA-Directed DNA Polymerase