摘要
目的 研制肝癌多种相关基因芯片 ,进行肝癌组织中基因编码区单核苷酸多态性(cSNP)检测分析研究。方法 选取定位于肝癌高频缺失区的基因 ,经NCBIdbSNP数据库查询 ,获得基因cSNP序列 ,且根据多态位点设计寡核苷酸探针 ,制备成含 2 5个肝癌相关基因的 4 8个cSNP芯片。并对 10例肝癌患者cSNP进行检测分析 ,而对 7例患者的部分检测结果用聚合酶链反应 单链构象多态性法 (PCR SSCP)和测序进行验证。结果 芯片检测的灵敏度为 6× 10 3 ng/ μl,检测重复率高于 95 83% ,随探针浓度降低 ,杂交信号逐渐减弱。 10份肝癌患者组织中 ,检测到半胱氨酸酶caspase9(rs2 30 894 1)C→T多态性和停泊蛋白DOK2 (rs2 2 4 2 2 4 1)T→G各 7份 ,表皮生长因子类似物EGFL3(rs94 7345 )A→G多态性、caspase9(rs2 30 8938)C→G多态性和磷酸甘油酸盐脱氢酶PHGDH(rs180 195 5 )T→A多态性各 6份 ,启动子结合因子E2F2 (rs32 18170 )G→A多态性 5份 ,DNA切除修复糖基化酶MUTYH(rs114 0 5 0 7)T→C和胞内蛋白BNIP3L(rs10 5 5 80 6 )G→T多态性 4份 ,肿瘤坏死因子家族成员TNFRSF1B (rs10 6 16 2 2 )T→G多态性 1份。经PCR SSCP检测 ,7例肝癌患者有多态性单链迁移率改变。选取有caspase9(rs2 30 894 1G)与 (rs2 30 894 1A)
Objective To prepare a gene chip for detecting polymorphisms on coding region of genes in hepatocellular carcinoma tissuses. Methods Genes harboring in loss regions with high frequency of hepatocellular carcinoma (HCC) were selected, the related information was inquired and the cSNP sequences were obtained in the SNP database of National Center for Biotechnology Information(NCBI).Then the appropriate primers and oligonucleotide probes were designed according to the SNP sites and the gene chip for the detection of SNPs were constructed. This chip includes 48 cSNPs of 25 hepatocellular carcinoma-related genes. The PCR products labeled by Dig-dUTP were hybridized with cSNP chip. Results The sensitivity, influence of probes and reiteration of the chip were detected. The results indicated that the chip was sensitive about 6×10^(-3) ng/μl. The signal of hybridization was down with lower concentrition of probes. By the chip, 7 of polymorphisms of caspase9 ( rs2308941) C→T and DOK2(rs2242241)T→G, 6 of polymorphisms of EGFL3(rs947345)A →G,caspase9 ( rs2308938)C→G and PHGDH(rs1801955)T→A, 5 of polymorphisms of E2F2(rs3218170) G→A,4 of polymorphisms of MUTYH(rs1140507)T→C and BNIP3L(rs1055806)G→T, 1 of polymorphism of TNFRSF1B (rs1061622)T→G were detected in the tissues of 10 HCC. Samples of caspase9 ( rs2308941G) and ( rs2308941A) were verified by PCR-SSCP and sequencing. Conclusion We prepare the cSNP chip of hepatocellular carcinoma-related genes, which can accelerate the discovery of polymorphic markers on hepatocellular carcinoma.
出处
《中华检验医学杂志》
CAS
CSCD
北大核心
2004年第10期649-652,共4页
Chinese Journal of Laboratory Medicine
