毛细管电泳法检测胃癌APC基因第十一外显子的杂合缺失

摘要

In this paper, the part base sequences of APC gene on exon 11 were extracted from normal tissue and gastric cancer tissue and amplified by polymerase chain reaction( PCR), which contains some bases easily resulting in loss of heterozygosis (LOH). Then the amplified samples were denatured at 96 ℃ and digested by restriction enzyme of Rsa Ⅰ and detected by capillary electrophoresis (CE) -single strand conformation polymorphism( SSCP), capillary electrophoresis -restriction fragment length polymorphism(RFLP) and polyacrylamide gel electrophoresis(PAGE) -SSCP. The concentration of polyacrylamide gel was selected to be 15％. The analysis of APC gene on exon 11 was performed by CE combined with laser-induced fluorescence detector(λex = 488 nm, λem = 520 nm),by using 3.0％ polyethylene oxide(PEO) as sieving medium, 1 × TBE ( pH 8.2) as electrophoresis buffer with a separation voltage of 15 kV at 15 ℃. The sequence of detection ratios for APC gene detected by different methods was as follows: CE -SSCP(30％) ＞ PAGE -SSCP(25％) ＞ CE -RFLP (20％). The results revealed that the LOH of APC gene in gastric cancer tissue was higher than 10％ , and the detection ratio for CE - SSCP was higher than that for PAGE - SSCP and CE - RFLP.Therefore, with the advantages of rapidness and high sensitivity, the CE - SSCP method could lay a good foundation for the convenient, reliable and early diagnosis on gastric cancer.%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Rsa Ⅰ酶切处理,以毛细管电泳(CE)-单链构象多态性(SSCP)、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.