| [Objective]Denaturing high-performance liquid chromatography (DHPLC) technology was applied to screen gene mutation in 1996 for the first time and since then, the technology has been widely accepted and exploited in basic research and clinical diagnosis mainly for it's sensitivity,accuracy,effectiveness etc. Following the technological investigation and development, the technology is also applied to multiple fields involving genome research besides the mutation detection, such as DNA microsatellite identification,tumor LOH detection, RF-PCR competitive quantification, genetic analysis of human race, gene mapping, research of nucleic acid self-excising, length analysis of DNA fragment, quality control of oligonucleotide and it's purification, analysis of mitochondrial DNA, analysis of CpG island methylationand so on. DHPLC technology has been broadly used abroad, both in laboratory and in clinic;there has been reports about use of the technology at home, but on the whole, the application is at the beginning and limited only in lab.As the third generation of molecular and genetic marker, single nucleotide polymorphism(SNP) is a most common inheritable alteration of human beings.lt is caused by a single base variation at genome level. Compared with the first and second generation of genetic marker, SNP has a advantage of high-density, stability and easily typing. As these reasons, SNP shows its huge predominance in the study of diseases, especially in polygene diseases. SNP has been used to study individual difference. Most SNPs are "silent", but at least part of them are related to the extent of disease development and severity.Wnt signaling pathway plays a pivotal role during some key physiological and pathological processes, such as embroygenesis, tumor initiation, etc. It is one of the most widely studied signal pathway and it has been demonstrated that the pathway participated in a variety of human tumor.Gastric carcinoma is one of the most common malignant tumors. The molecular mechanism is very complex, which includes alterations of oncogene, tumor suppressor gene and tumor-related gene. There has been many research about gastric carcinoma-related genes, and some SNPs locating in their promoters have been demonstrated to be associate with the initiation and development of gastric carcinoma. Howerer, whether SNPs in encoding gene of Wnt signal pathway components are related to the risk of gastric carcinoma or not, there has no corresponding report. Based on the above description, the study was carried out to analyze the allele frequencies and genotypes of four SNP sites locating in encoding gene of Wnt signal pathway components: SNP rs3755557 in glycogen synthase kinase 3/3(GSK-3/3) gene, SNP rsl 880481 and rs3864004 in (3-catenin gene, SNP rs3777860 in adenomatous polyposis coli(APC) gene. The study aimed to discuss correlation between these SNPsand gastric carcinoma. [Methods]According to the experiment design, subjects were divided into two groups—control group (gastritis group) including 61 patients of gastritis and gastric carcinoma group including 26 patients of gastric carcinoma. The experimental process was as follows: Genomic DNA was purified from human leukocytes by SDS/proteinase Kdigestion and phenol extration.;four pairs of oligonucleotides were designed to amplify the target DNA fragments;appropriate sized PCR reaction products were confirmed by agarose gel electrophoresis and DNA sequencing;PCR product was cycled by renaturing and subsequently reannealing to form homoduplex and heteroduplex, and then it was genotyped though DHPLC technology in combination with DNA sequencing. The detection process by DHPLC technology was as follows: a sample of heteroduplex peak and another sample of homoduplex peak were sequenced to identify their genotypes;the sample that presented homoduplex peak was mixed with others;the mixture was hybridized as the previously described method, then it went through DHPLC detection process. Homoduplex chromatogram of this test result meant the two samples in the mixture had the same genotype and they were both homozygous;heteroduplex chromatogram meant the two sample were different and the unknown sample was another homozygous.[Results]The following results were obtained through the study: (l)As to the SNP sites rs3755557, rsl88048land rs3777860, double peaks and single peak corresponded clearly to heteroduplex and homoduplex chromatogram;the results of DNA sequencing and DHPLC detection were identical. (2) Chi-square analysis revealed that there was no significant difference in the frequencies of alleles and genotypes ofrs3755557 and rs3777860 polymorphic site between gastric carcinoma group and control group. (3) As to the rsl 880481 polymorphic site, there was no significant difference between gastric carcinoma groups and the corresponding control groups. The frequency of heterozygous genotype in male control group was 59.52% and this was significantly higher than 26.67% in male gastric carcinoma group, OR=0.247, 95%CI: 0.067~0.907(P=0.038). (4) The ideal detection result of rs3864004 site was not obtained.[Conclusion](1) The genotypes and alleles frequencies of rs3755557 site of GSK-3/5 gene and rs3777860 site of APC gene do not contribute to the risk of gastric carcinoma. (2) Low level of the heterozygous genotype of rsl 880481 polymorphic site of /3-catenin in male patients might cause a higher risk of developing gastric carcinoma. (3) DHPLC technology is a effective and accurate technology to detect SNP.
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