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/?l. 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.
ABSTRACT
Objective To study the preparation of hepatitis C viruses (HCV) genotyping oligochip and its application in the detection of 76 hepatitis C patients.Methods Oligonucleotide probes and primers were designed in the 5’noncoding region and core region of HCV. The HCV typing chip was prepared by spotting the modified probes onto nylon membrane. Products of the second PCR were labeled with Dig-dUTP. Furthermore, 6 PCR products were sequenced.Results Using the chip,15 subtypes in 11 types of HCV were analyzed.Results of hybridization indicates that 76 hepatitis C patients were all positive and 20 health people were negative.Among 76 patients, 64 cases were 1b type, 11 cases were 2a type and 1 case was 3a type. Mix infection was not found. The results obtained by sequencing 6 samples and chip arraying were the same.Conclusion The HCV genotyping chip could be used in detecting serum HCV RNA and analyzing its genotypes.
ABSTRACT
Objective To analyze the DNA methylation status of polyploid complex of Pinellia ternata. Methods Methylation sensitive amplified polymorphism(MSAP) technique was carried out to analyze the methylation status of polyploid complex of P.ternata. Thirty-four selective primer amplifications were used to check the status of cytosine methylation DNA samples. Results A total of 7 708 bands were obtained.Among them 5 636 bands,each representing a recognition site cleaved by one or both of the isoschizomers(Hpa Ⅱ and Msp Ⅰ),were amplified.Furthermore,methylation patterns varied among the four polyploids:heptaploid,octoploid,nonuploid,and decaploid.Total and full methylation levels in P.ternata were 54%-58% and 24.1%-24.3%.All types of MSAP patterns detected in the study belonged to two classes,type Ⅰ and Ⅱ. 52.5% of detected bands belonged to Type Ⅰ; Another 47.5% were type Ⅱ,which showed the methylation differences among the four polyploids. Conclusion The results demonstrate DNA methylation events occur in P.ternata and the general methylation levels are higher.