Abstract
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.
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