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目的:应用基因扫描法检测人表皮生长因子受体(EGFR)基因19外显子缺失突变。方法:(1)将EGFR基因19外显子缺失(del E746-A750)型及野生型基因分别克隆到T载体,并将含突变型基因及含野生型基因的载体按1:1、1:5、1:10、1:50、1:100、1:300的比例进行混合,用基因扫描法检测19外显子的缺失,以确定其灵敏度。(2)基因扫描法检测225例非小细胞肺癌患者的手术组织或穿刺标本;荧光标记EGFR基因19外显子的上游引物,PCR扩增目的片段,3100A测序仪进行基因扫描,Gene Scan Genotyper 3.5软件分析结果。同时用直接测序法检测EGFR19外显子的缺失突变。结果:(1)基因扫描法可测出1:100混合液中的突变型,灵敏度较高。(2)225例肺癌标本,可检测出27例(12.0%)缺失;直接测序法则检出25例(11.5%)缺失突变。结论:基因扫描法检测EGFR基因19外显子的缺失,方法简便,成本低廉,敏感度高,可查出1%的突变。
OBJECTIVE: To detect mutations in exon 19 of the human epidermal growth factor receptor (EGFR) gene by gene scanning. Methods: (1) The EGFR gene exon 19 (del E746-A750) and wild type genes were cloned into the T vector, and the vector containing the mutant gene and wild type gene was 1:1 and 1: Mix at a ratio of 1:10, 1:10, 1:100, and 1:300. Detect the deletion of exon 19 by gene scanning to determine its sensitivity. (2) Gene scanning method to detect surgical tissue or puncture specimens of 225 patients with non-small cell lung cancer; Fluorescence labeling of upstream primers of exon 19 of EGFR gene, PCR amplification of the target fragment, gene scanning on the 3100A sequencer, Gene Scan Genotyper 3.5 Software analysis results. Simultaneously, direct sequencing was used to detect deletion mutations in EGFR19 exons. Results: (1) The gene scanning method can detect mutants in a 1:100 mixture with high sensitivity. (2) In 225 lung cancer specimens, 27 (12.0%) deletions were detected; in direct sequencing, 25 (11.5%) deletion mutations were detected. Conclusion: The gene scan method was used to detect the absence of exon 19 of EGFR gene. The method is simple, low-cost, and sensitive. It can detect 1% of mutations.