毛细管电泳法检测癌基因C-myc胃癌中基因点突变

摘要

癌基因C-myc激活和突变在胃癌形成过程中起着重要作用.通过毛细管电泳(CE)方法检测50例胃癌患者中C-myc基因突变,建立一种准确、快速诊断早期胃癌的方法.本实验采用PCR扩增胃癌及癌旁正常组织中C-myc基因第二外显子易发突变的部位基因序列,扩增样品分别经96℃变性和EcoRV酶切处理,以PAGE-SSCP,CE-SSCP,CE-RFLP分别对其突变情况进行检测.优化的CE检测条件:筛分介质PEO浓度3.0％,pH 8.2,电压15 kV,温度15℃；荧光检测:λex=488 nm,λem=520 nm.检测结果:C-myc基因总突变率为20.0％ (10/50).测序分析结果显示C-myc基因第二外显子第53密码子存在点突变,碱基A变为碱基T(GAT→GTT),碱基的改变使氨基酸由亮氨酸替代为谷氨酰胺.本研究数据证实C-myc基因突变与胃癌的形成紧密相关,CE检测C-myc突变基因可作为胃癌早期诊断的简便可靠的方法.%Activation of C-myc oncogene by mutation has been reported to play an important role in gastric cancer tumorigenesis. We determined C-myc mutation in 50 patients with gastric cancer by capillary electrophoresis (CE) to establish an exactly and volant clinical diagnostic method in early gastric cancer. In this experiment, genomic DNA was extracted from normal tissue and gastric cancer tissue and the sequence of C-myc oncogene on exon 2 (possible with high mutation frequency) in the extracted genomic DNA was amplified by polymerase chain reaction (PCR). Then amplified DNA samples were denatured at 96 oC and digested by EcoRV and detected by PAGE-single strand conformation polymorphism (SSCP), CE-SSCP, CE-restriction fragment length polymorphism (RFLP). The optimum CE detection conditions including 3. 0% sieving medium poly(ethylene oxide) (PEO), pH 8. 2, separation voltage of 15 kV and temperature of 15 oC were adopted. The laser-induced fluorescence detector was set at λex = 488 nm, λem = 520 nm. The results reveal that the mutation frequency of C-myc gene is 20. 0% (10/50). Sequence analysis further identified a point mutation of A to T in exon 2 codon 53 (GAT→GTT), causing an amino acid substitution of leucine to glutamine. This study may suggest a correlation of the mutation of C-myc oncogene with gastric cancer progression. This work also demonstrates that CE can offer a convenient and reliable method to early diagnosis of gastric cancer.