摘要
目的:建立多个基因同时进行多态性分析的序列特异性引物多聚酶链反应的方法,以满足其在临床检验中进行基因多态性鉴定时的应用。方法:实验于2005-10/2006-05于河南省新乡医学院分子生物研究室完成。进行临床普通血液常规检测的患者血样200份(由新乡医学院第三附属医院提供),采用临床血样,提取DNA后采用多聚酶链反应扩增,并以扩增后的样品进行琼脂糖凝胶电泳,以是否出现相应的扩增条带作为基因分型的标准。应用优化后的序列特异性引物多聚酶链反应的方法分析以下基因的多态性:肿瘤坏死因子α-308A/G和-238G/A变异、白细胞介素6-174G/C变异、细胞色素P4502D6(CYP2D6)*10B外显子第188位的C/T变异。并由此优化序列特异性引物多聚酶链反应的方法,以达到应用同一个多聚酶链反应扩增程序,可同时对多个基因、多个临床样本的基因多态性同时进行分析,且基因型清晰,分型快速、准确。结果:①用20 g/L的琼脂糖凝胶电泳可看到每泳道内可有2条或1条扩增产物,其中阳性对照β珠蛋白基因的扩增片段长268 bp,此条带可以出现、减弱或者不出现。②其中肿瘤坏死因子α-308A/G存在G/G和A/G两种基因型;肿瘤坏死因子α-238G/A位点可检测到的基因型有G/A、G/G和A/A3型;白细胞介素6-174G/C变异位点可检测到的基因型均为G/G纯合子;细胞色素P4502D6*10B外显子第188位的C/T位点可检测到的基因型为C/C、C/T、T/T3型。结论:优化后的序列特异性引物多聚酶链反应适合对大样本,多个基因的单核苷酸位点变异进行多态性分析,快速、准确、成本低。
AIM： To establish the method of sequence specific primers polymerase chain reaction （PCR-SSP） procedure to identify polymorphisms for multiple genes simultaneously, and satisfy the application of gene polymorphism evaluation in the clinical checks.
METHODS： The experiment was conducted in the Department of Molecular Organism, Xinxiang Medical College between October 2005 and May 2006, Samples contained 200 patients were recruited from the Third Affiliated Hospital of Xinxiang Medical College and then detected routinely. After the genomic DNA extraction, samples were amplified with PCR and analyzed with agarose gel electrophoresis （AGE） to determine the genotype through the visibility of amplification band. The optimized PCR-SSP method was used to analyze the polymorphisms of following genes： tumor necrosis factor （TNF） α-308A/G, TNFα-238G/A, interleukin （IL） 6-174G/C, CYP2D6 ^＊10B extron 188C/T. Then it was modified to optimize polymorphism analysis for multiple genes and many clinical samples could be performed simultaneously with one PCR amplification program; showing clear genotype, quick and accurate genotyping.
RESULTS： ①AGE of 20 g/L was used to demonstrate one or two amplification bands visible in one lane, positive control was the 268bp fragment of β globin gene, and the band was present, attenuated or not present.②For the TNFα-308A/G locus, two genotypes （GIG and A/G） were detectable; For TNFα-238G/A site, three genotypes （G/A, GIG and A/A） were detectable; For the IL-6-174G/C locus, only the homozygote G/G was detectable; For the C/T locus at 188 in exon of CYP2D6^＊ 10B, three genotypes （C/C, C/T and T/T） were detectable.
CONCLUSION： The optimized PCR-SSP is suitable for pofymorphlsm analysis of the large-sample polygenic mononucleotide site mutation, and It can be widely applied in clinical test for low cost, quickness and accuracy.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2007年第2期321-324,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
省教育厅资助项目(200510472002)~~
