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采用聚合酶链反应(PCR)扩增了胃癌及癌旁正常组织中APC基因易发生杂合缺失的第十一外显子的部分碱基序列,扩增样品分别经96℃变性和RsaⅠ酶切处理,以毛细管电泳(CE)-单链构象多态性(SS-CP)、CE-限制性片段长度多态性(RFLP)、聚丙烯酰胺凝胶电泳(PAGE)-SSCP对其杂合缺失情况进行检测。PAGE凝胶浓度为15%。CE检测条件:以3.0%聚环氧乙烷(PEO)为筛分介质,1×TBE(pH 8.2)为电泳缓冲液,分离电压15 kV,温度15℃,结合激光诱导荧光(λex=488 nm,λem=520 nm)检测。不同方法的检出率由高到低分别为:CE-SSCP(30%)>PAGE-SSCP(25%)>CE-RFLP(20%)。并证实了APC基因易发生杂合缺失在胃癌组织中的突变率高于10%,且CE-SSCP方法较PAGE-SSCP和CE-RFLP的检出率高。CE-SSCP检测方法具有快速、灵敏度高等优点,可为建立简便可靠的胃癌临床早期诊断方法奠定基础。
The polymerase chain reaction (PCR) amplification of gastric cancer and adjacent normal tissue APC gene is prone to heterozygous deletion of eleven exons of partial base sequence, amplified samples were 96 ℃ denaturation and Rsa Ⅰ digestion (SS-CP), CE-restriction fragment length polymorphism (RFLP) and polyacrylamide gel electrophoresis (PAGE) The situation is tested. PAGE gel concentration of 15%. CE detection conditions: 3.0% polyethylene oxide (PEO) was used as the screening medium, 1 × TBE (pH 8.2) was used as electrophoresis buffer, the voltage was 15 kV and the temperature was 15 ℃. Combined with laser induced fluorescence (λex = 488 nm , λem = 520 nm). The highest and lowest detection rates of different methods were: CE-SSCP (30%)> PAGE-SSCP (25%)> CE-RFLP (20%). And confirmed that heterozygous deletion of APC gene was more than 10% in gastric cancer. The detection rate of CE-SSCP was higher than that of PAGE-SSCP and CE-RFLP. The CE-SSCP detection method has the advantages of fastness and high sensitivity, which can lay a foundation for establishing a simple and reliable method for clinical early diagnosis of gastric cancer.