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随着基因组关联分析方法的应用,越来越多与胃癌相关的易感基因被发现.易感基因的多态性检测已逐步进入胃癌临床诊断和研究.然而,利用少量胃粘膜细胞开展单核苷酸多态性(SNP)分析对胃癌进行早期诊断常遇下述困难,一是少量胃癌细胞混杂在多种细胞中,异常信号常易被淹没,二是细胞量极少,因此获得的基因组DNA量微,进行多位点或全基因组分析存在困难.本文利用激光显微切割技术分选少量胃癌细胞,结合全基因组放大技术,进行胃癌相关的前列腺干细胞抗原基因(PSCA)的SNP分析.通过聚合酶链反应-限制性片段长度多态性(PCR-RFLP)和克隆测序方法分析,在分选的胃癌细胞中检测到PSCA的rs2976392位点胃癌相关的“A”等位与rs2294008位点胃癌相关的“T”等位.研究结果表明,所采用的全基因组放大方法保真性高,经过分选的胃癌细胞中SNP位点的检测灵敏度和可靠性大为提高.所建立的少量细胞基因多位点检测方法将同样应用于其它肿瘤和组织的少量细胞研究中,全基因组放大产物也可进行高通量的基因芯片和第二代测序研究.
With the application of genomic association analysis, more and more susceptible genes associated with gastric cancer have been found.Polymorphism of susceptible genes has gradually entered the clinical diagnosis and research of gastric cancer.However, the use of a small amount of gastric mucosal cells to carry out mononuclear Analysis of nucleotide polymorphisms (SNP) often leads to the following difficulties in the early diagnosis of gastric cancer. First, a small amount of gastric cancer cells are mixed in a variety of cells. Abnormal signals are often submerged. Second, the amount of cells is very small. Therefore, the obtained genome It is difficult to carry out multi-locus or genome-wide analysis of DNA.We used laser microdissection to classify a small number of gastric cancer cells and combined SNP analysis of gastric cancer-associated prostate stem cell antigen gene (PSCA) Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and cloning sequencing analysis showed that gastric cancer-associated “A” allele of rs2976392 in PSCA was detected in sorted gastric cancer cells with rs2294008 Point “T ” alleles associated with gastric cancer.The results show that the genome-wide amplification method used has high fidelity and greatly improves the detection sensitivity and reliability of SNP loci in sorted gastric cancer cells A small amount of cellular gene detection method multiple established cell research will be equally applicable to other tumor and a small amount of tissue, the whole genome amplification product can also be a gene chip and a second-generation high-throughput sequencing studies.