关键词: 基因定点突变, 截短基因, 双片段连接, 周期蛋白E

Abstract: The random mutation associated with DNA synthesized in vitro is an important factor that restricts the efficiency of site-directed mutation. Taking cyclin E and its truncated genes as an example, in order to clone their kinase-deficient mutants, the traditional overlap extension PCR (OE-PCR) was improved appropriately, and an improved scheme about site-directed mutagenesis for series related genes was proposed. In the previous study, cyclin E gene and its two truncated genes T1 and T2 were cloned and inserted into pEGFP_C2 vector by EcoR Ⅰ/Sal Ⅰ double digestion. Restriction analysis showed that cyclin E gene sequence contained one Age Ⅰ site, which was divided into F1 and F2 segments, and the target mutation site KD was located in F2 segment. The F2 segment was identical to cyclin E and its truncated gene. Therefore, the consensus fragment F2 containing target mutation was amplified by overlap extension PCR (OE-PCR), and the corresponding F1 fragments were cut out from the three original plasmids C2-cyclin E, C2-cyclin E_T1 and C2-cyclin E_T2, and then F1 and the enzyme digested F2 were ligated into the vector to reconstruct the complete mutant. Comparative detection showed that OE-PCR was more successful in amplifying shorter DNA fragments. A certain number of clones were screened from C2-cyclin E, C2-cyclin E_T1 and C2-cyclin E_T2. After detection and DNA sequencing, one target mutant with completely accurate sequence was obtained from each group. Studies have shown that partial amplification could shorten the length of DNA synthesis, avoid the unfavorable factors such as repeated amplification of target genes, and thus reduce random mutations. And double-fragment ligation avoided the restriction of double Age Ⅰ sites on routine restriction-ligation. The combination of the two methods could be used as an optimization scheme for site-directed mutation of other related genes.

Key words: site-directed mutagenesis, truncated gene, double fragments ligation, Cyclin E