A direct and efficient PAGE-mediated overlap extension PCR method for gene multiple-site mutagenesis.
A simple, two-step efficient method to perform multiple-site mutagenesis of a gene from bacterial genome was developed. The method was named polyacrylamide gel electrophoresis (PAGE)-mediated overlap extension polymerase chain reaction (PCR) (POEP). The first step involves synthesis of individual fragments containing mutant sites with 15- to 25-bp overlap between two adjacent fragments. Mutations were introduced into the overlapping oligonucleotide primers which ensured the particular primer-template annealing. PAGE was used to remove contaminating parental templates, mispriming fragments, and leftover primers. The second step involves synthesis of the mutant full-length fragment. All purified PCR products from the first step were combined and used as the template for a second PCR using high-fidelity DNA polymerase, with the two outermost flanking oligonucleotides as primers. Using the POEP method, we have successfully introduced eight EcoRI sites into the Escherichia coli beta-galactosidase (Lac Z) gene. The overall rate of obtaining the multiple mutant sites was 100%. The POEP method is simple, involving only two steps, and reliable for multiple-site mutagenesis and is promising to be widely used in gene modification.
Peng
Ri-He
RH
Shanghai Key Laboratory of Agricultural Genetics and Breeding, Agro-Biotechnology Research Center, Shanghai Academy of Agricultural Sciences, 2901 Beidi Rd, Shanghai, People's Republic of China.
Xiong
Ai-Sheng
AS
Yao
Quan-Hong
QH
D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D015246:Deoxyribonuclease EcoRI / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D008967:Molecular Biology / Q000379:methods*; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D001616:beta-Galactosidase / Q000235:genetics
D004269:DNA, Bacterial; D015246:Deoxyribonuclease EcoRI; D001616:beta-Galactosidase