| Human Genome Project ( HGP) displays us the vast foreground in human genetics. Cloning and location of pathogenetic genes including tumor and other diseases are one of the major contents in human Genome Project. Because of the advantage of patients'resources in our country, cloning and location of pathogenetic genes according to our developmental status of genetics are of hot spots. Tumurigenesis is a complex multi - stages process in which multi - factors and multi - genes are involved. Therefore, the detection of cancer ?related genes in different tumors is the basic prerequisite to understand the mechanism of turnori-genesis and to conduct gene diagnosis and gene therapy. Kundson " two hit" hypothesis and Cavanee "loss of heterozygosity (LOH)" concept indicate the relationship between allelic loss and Tumurigenesis, and locate LOH on chromosome in the level of DNA. According to the theories, Allelic loss of a particular chromosomal region in a tumor is thought to show that a tumor suppressor gene ( TSG ) normiilly resident there has been deleted. Rbl APC DCC WF1 BRCA1 BRCA2 genes have been cloned successively in this kind of strategy. Therefore, it is one of hot spots in tumor molecular genetics to identify TSGs by mapping regions of LOH. Researchers are minifying regions of LOH where TSGs are harbored for cloning one or more TSGs. Such as scholars in our country discovered HCCSI, a 2. 0 kb novel candidate of TSG containing 18 exons in region of LOH on chromosome 17pl3.3 (D17S5 -D17S34).Gastric cancer ( GC) is one of the most common humon malignancies and remains an important cause of mortality in China. At present, many regions of LOH on different chromosomes have been found in GC. These TSGs and candidate TSGs involved in GC include p53 Smad4 p16 DCC and so on . Some newputative tumor suppressor sites have been identified successively in GC. Therefore, the urgent matter of the moment is discovering continuously new sites of LOH and mapping commonly deleted regions on chromosomes in GC. Further narrowing the regions of frequent LOH will be required to map and clone new critical TSGs involved in the development of GC, and inquiry into its biology function.Based on previous results of comparative genomic hyhridization ( CGH) , we conducted more extensive a genome - wide allelotyping on chromosomes 17 and 18 using highly polymorphic microsatellite markers, and identified the chromosomal sites and overlapping regions of frequent deletion. It is useful to clarify the critical role of LOH on chromosomes 17 and 18 in GC and to provide evidences for discovering new TSGs.Materials and methods1. Samples collection45 surgically resected primary gastric tumors and corresponding nontumor-ous tissue specimens were obtained from the firsts the second and the third affiliated hospital of Harbin Medical University and stored at - 80c. All the patients were confirmed by routine histologic examination and received no treatment before surgery.2. DNA extractionDNA extraction was performed according to the protocol described in DNAzo1R reagent - genomic DNA isolation reagent manuals. The concentration and purity of DNA stocks were estimated by pharmacia DNA/RNA calculator.3. Choice label and PCR reaction system of the first batch primersOut of the entire chromosome 17, we chose 15 highly polymorphic microsatellite markers (7 markers at 17p, 8 markers at 17q) at a density of one marker every 8cM on average; Out of the entire chromosome 18, we chose 14 highly polymorphic microsatellite markers (5 markers at 18p, 9 markers at 18q) at a density of one marker every 9cM on average for allelotyping in 45 primary GC patients with lower - density microastellite markers. The oligonucleotites werelabeled with three different fluorescent dyes FAM^HEX and NED (primers were obtained from the ABI prism linkage mapping set v. 2, Perkin -Elmer). Multiplex PCR (4-15 primers) was carried out in a Gene Amp PCR system 9600 (Perkin - Elmer) for amplifying matched pairs of normal and rumor DNAs. PCR rea...
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