PCR-双酶切鉴定STGC3抑癌基因重组子假阳性研究

Analyzing the False Positive of PCR and Double Enzyme Digestion in Identifying STGC3 Suppressor Gene Recombinant

目的:探讨PCR与双酶切技术联合使用鉴定阳性重组子的特异性和可靠性,评价长期保存的重组质粒再次测序的必要性。方法:对随机选取已经过测序验证的三组STGC3/pcDNA 3．1(+)、一组STGC3/pGEM Easy T Vector重组子首先经过LB氨苄平皿培养挑单克隆筛选,然后单独或联合使用PCR和双限制性内切酶BamH I和Xho I酶切方法鉴定,将阳性样品送交生物工程公司测序,比较并评价两者结果的一致性。结果:三组STGC3/pcDNA 3．1(+)重组子经PCR—双酶切检测阳性而不能通过测序,说明该检测方法在运用中确实存在假阳性,同时证明重组载体在长期保存后有必要再次鉴定。结论:保存过程中的杂菌污染、实验室操作中的质粒交叉污染以及重组载体基因突变是基因工程操作中不可忽视的问题;PCR—双酶切鉴定重组载体存在假阳性的问题,实验设计中应包含阳性与阴性(污染)对照,并有足够的重复实验。

Objective： Recombinants are playing a very important role in gene engineering, PCR and double enzyme digest are key techniques to verify the positive recombinants, both of which are also liable to create false positive results, Otherwise, the long-time keeped recombinants might need to be rechecked whose accuracy structures might be changed under some conditions. In addition, PCR-Double Enzyme Digestion and sequencing methodologies were discussed including sensitivity requirements, applying prospect, cross-contamination problems, tissue sampling procedures and good laboratory practice （GLP） compliance, Methods Single or both of PCR and double digest techniques by BamH Ⅰ and Xho Ⅰ restriction enzyme digestion were used to assess four STGC3 gene recombinants which were screened by LB AMPr culture first. Empty vector was acted as positive control. Then all samples were examined with the sequencing consequences. Results： Three STGC3/pcDNA3.1 （＋） recombinants were failure to pass the sequencing which were not accordance to previous outcomes of PCR and double enzyme digestion. The STGC3/pGEM T vector recombinant and control vectors were with one accord. Conclusions： False positive was verified although two techniques had been extensively using in justifying positive recombinants and rechecking recombinants in stock might be necessary. Three main causes are bacterial contamination, laboratory-generated plasmid contamination and gene mutants. So positive control and negative control （as control for contamination） are much needed in parallel with each batch of samples as well as adequate repeats.