用DREAM技术进行全长质粒快速定点突变

Rapid site-directed mutagenesis on full-length plasmid DNA by using designed restriction enzyme assisted mutagenesis

利用"设计限制酶辅助突变"(Designed Restriction Enzyme Assisted Mutagenesis,DREAM)进行全长质粒快速定点突变。根据突变位点附近氨基酸靶序列,以简并密码子进行逆向推导,这样在不改变氨基酸序列的前提下可以得到数目巨大的隐性突变体(Silent mutants),这些突变体中包含大量的限制性酶切位点,选择合适的酶切位点设计引物,用Phusion超保真DNA聚合酶扩增全长质粒的DNA序列,得到的PCR产物用T4多聚核苷酸激酶添加5′磷酸基团后进行平末端连接,转化大肠杆菌受体菌后用设计的酶切位点进行快速筛选。本研究用该方法成功地纠正了长约8 kb的质粒pcDNA3.1-pIgR中的突变碱基,从而获得了多聚免疫球蛋白受体(pIgR)的野生型氨基酸序列。以上结果表明:利用DREAM技术将限制性酶切位点引入目的基因而不改变目的蛋白质的氨基酸序列,使突变体的筛选简单化;配合使用高保真和高效率的Phusion DNA聚合酶可以进行长达8 kb的全长质粒的快速突变;该方法无需使用定点突变试剂盒和特殊的受体菌,同时避免了核酸杂交以及同位素的使用。

To use the designed restriction enzyme assisted mutagenesis technique to perform rapid site-directed mutagenesis on double-stranded plasmid DNA. The target amino acid sequence was reversely translated into DNA sequences with degenerate codons resulting in large amount of silently mutated sequences containing various restriction endonucleases （REs）. Certain mutated sequence with an appropriate RE was selected as the target DNA sequence for designing mutation primers. The full-length plasmid DNA was amplified with high-fidelity Phusion DNA polymerase and the amplified product was 5＇ phosphorylated by T4 polynucleotide kinase and then self-ligated. After transformation into an E.coli host the transformants were rapidly screened by cutting with the designed RE. With this strategy we successfully performed the site-directed mutagenesis on an 8 kb plasmid pcDNA3, l-pIgR and recovered the wild-type amino acid sequence of human polymeric immunoglobulin receptor （pIgR）. A novel site-directed mutagenesis strategy based on DREAM was developed which exploited RE as a rapid screening measure. The highly efficient, high-fidelity Phusion DNA polymerase was applied to ensure the efficient and faithful amplification of the full-length sequence of a plasmid of up to 8 kb. This rapid mutagenesis strategy avoids using any commercial site-directed mutagenesis kits, special host strains or isotopes.