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目的:构建了29种人类EGFR基因突变体质控品,为人类EGFR基因突变检测试剂盒的性能评价提供了可靠的质控物质。方法:根据人类EGFR全基因序列,应用Primer Premier 5.0软件在选取的序列区域设计引物,通过PCR介导的定点突变技术引入突变位点、PCR产物与克隆载体连接,转化感受态细菌,筛选阳性克隆等,建立了包含不同人类EGFR基因突变体的质控品。结果:对29种人类EGFR基因突变体质控品进行序列测定,并将测序结果进行BLAST比对,结果表明成功建立了人类EGFR基因突变体质控品。结论:本研究建立的EGFR基因突变体质控品涵盖了EGFR基因的29种突变位点,覆盖了目前最常见的EGFR基因突变类型,能够用于评价国内大多数EGFR基因突变检测试剂盒。
OBJECTIVE: To construct 29 kinds of human EGFR gene mutants and to provide a reliable controllable substance for evaluating the performance of human EGFR gene mutation detection kit. Methods: Primer Premier 5.0 software was used to design primers in the selected region of the sequence according to the human EGFR gene sequence. The site of mutation was introduced by PCR-mediated site-directed mutagenesis. The PCR product was ligated with the cloning vector and transformed into competent bacteria to screen positive clones Etc., have been established containing different human EGFR gene mutant quality control. Results: Sequencing of 29 human EGFR gene mutants was carried out. The results of BLAST analysis showed that human EGFR gene mutants were successfully established. CONCLUSIONS: The EGFR gene control plasmid contained in this study covers 29 EGFR gene mutation sites and covers the most common EGFR gene mutation types. It can be used to evaluate most EGFR gene mutation detection kits in China.