利用DREAM设计和同源重组进行一步定点突变

目的：建立基于DREAM设计和同源重组的简便、快速定点突变方法。方法：设计两条包含突变的反向PCR（inverse PCR）引物，使其5'端互补从而产生同源重组，同时使用DREAM设计方案在上述引物中引入限制性内切酶位点以便突变子筛选。用能扩增长片段的高保真耐热 DNA聚合酶扩增全长的质粒DNA，直接转化大肠杆菌。转化到细菌中的全长质粒DNA PCR产物可利用其末端同源序列发生同源重组而环化。利用引入的酶切位点方便地进行突变子的筛选。结果：我们用该方法成功地对长度大于7 kb的质粒进行了定点突变。结论：本定点突变无需任何突变试剂盒和特殊的试剂，只需一步反应即可完成；利用DREAM设计使克隆筛选简便可靠，高保真耐热DNA聚合酶可保证多数突变子克隆不发生意外突变，而该酶扩增长片段的能力使该方法适合于大多数质粒不经亚克隆直接突变。

One-step site-directed mutagenesis based on homologous recombination and DREAM design

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.