Mutagenesis by Overlap Extension

목차

1. Materials
2. Method
3. References

본문내용

Overlap extension PCR was developed almost concurrently by Higuchi and associates and by Ho and colleagues. (Interestingly, most subsequent papers only cited Ho et al.). Two PCRs are performed to produce two DNA fragments that carry overlapping sequences (Fig. 1C) and intended mutations, both of which are introduced by two mutagenic middle primers. Two PCR fragments are then mixed, denatured, and annealed in a PCR-ready buffer, to generate two heteroduplexes via overlapping sequences. Only one of the two heteroduplexes, which carries two 3‘-termini at the joint, can be extended by Taq DNA polymerase to form a full-length double-stranded mutant DNA. The other heteroduplex, since it carries two 5’-termini at the joint, is not extendible. The extended double-stranded mutant DNA is amplified in a further PCR using two outside primers.

FIG. 1. Introducing mutations into the middle of a DNA (C) Introducing mutations by in vitro overlap-extension PCR. Two PCRs are performed to produce two fragments that carry overlapping sequences. These two fragments are then mixed, denatured, and annealed in a PCR-ready buffer to obtain mutant DNAs in a further PCR.

참고 자료

Sambrook and Russell, Molecuar cloning 3rd, volume 2 : 13.36~13.39
Ling MM, Robinson BH. Approaches to DNA mutagenesis: an overview.
Anal Biochem. 1997 Dec 15;254(2):157-78. Review.
Michael, S. F. (1994) BioTechniques 16, 410–412.
Higuchi, R., Krummel, B., and Saiki, R. K. (1981) Nucleic Acids Res. 16, 7351–7367.
Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K., and Pease,L. R. (1989) Gene 71, 51–59.