中国生物工程杂志

To develop a rapid and convenient site-directed mutagenesis method based on homologous recombination and Designed Restriction Endonuclease Assisted Mutagenesis (DREAM). Methods: Two inverse PCR primers containing the desired mutation were designed with complimentary 5' sequences for homologous recombination and a restriction site introduced by DREAM design for rapid mutant screening. Full-length plasmid DNA was amplified with the above primers with a high-fidelity thermostable DNA polymerase capable of long-rang amplification. The amplified full-length plasmid PCR products were transformed into competent E. coli and they were able to circularize due to the terminal homologous sequences. The mutants were screen by convenient restriction digestion. Results: With this strategy we successfully performed the site-directed mutagenesis on a plasmid over 8 kb in length. Conclusion: This site-directed mutagenesis method contains only one step, and no mutation kits or any special regents are needed. Introduction of a restriction site enables convenient and reliable mutant screening. The use of a high-fidelity thermostable DNA polymerase guarantees that most of the mutants are free of unwanted mutations and its capacity of long-rang amplification makes this method applicable to most plasmids.