RsaⅠ与SmaⅠ多态性寡核苷酸阵列检测法的建立及其与血栓性疾病的相关性研究  

The Rsa Ⅰ and Sma Ⅰ polymorphisms in the von Willebrand factor gene discrimination by hybridization with oligonucleotide arrays

作　　者：戴克胜[1] 赵雨杰[2] 王宏 高维强[1] 王迎春[1] 阮长耿[1] 

机构地区：[1]苏州大学附属第一医院 [2]东南大学吴健雄实验室

出　　处：《中华医学杂志》2001年第19期1153-1156,共4页National Medical Journal of China

基　　金：国家自然科学基金资助项目 ( 39830 1 80 )

摘　　要：目的　建立血管性血友病因子 (vWF)基因RsaⅠ、SmaⅠ多态性寡核苷酸阵列检测方法 ,对基因芯片在单核苷酸多态性 (SNP)检测上的应用进行探讨 ;研究两位点多态性与血栓性疾病的发生有无相关关系。方法　设计、合成两组寡核苷酸探针 ,使用 3甲氧基氨基丙基硅烷、戊二醛等化学物质 ,实现探针与固相支持物玻片的连接。应用不对称PCR方法扩增RsaⅠ、SmaⅠ多态性片段 ,在扩增体系中掺入荧光标记dUTP ,获得被测片段的单链标记产物。对核酸杂交反应的温度、动力学和离子浓度变化进行研究 ,获得最佳的杂交鉴别条件。对 2 0名正常人 ,酶切法确定多态性基因型 ,再用寡核苷酸阵列法检测 ,以验证该方法的准确性。使用该方法对 50例血栓病人进行检测 ,探讨血栓性疾病的发生与vWF基因RsaⅠ、SmaⅠ多态性的关系。结果　寡核苷酸阵列法和酶切法对 2 0例标本检测的符合率为 1 0 0 % ;血栓病人与正常人RsaⅠ、SmaⅠ两位点多态性的基因型GG、GA、AA和CC、CT、TT分别为 4.0 %、1 2 .0 %、84.0 %和 2 4.0 %、44.0 %、32 .0 % ,正常人分别为 1 .4%、1 1 .8%、86 8%和 8.8%、57.4%、33 8% ,血栓病人等位基因频率G、A和C、T分别为 1 0 .0 %、90 .0 %和 46 0 %、54 .0 % ,正常人分别为 7.4%、92 .6 %和 37 5%、6 2 .5% ,血栓病人?Objective To develop a new assay for the Rsa Ⅰ and Sma Ⅰ polymorphisms (single nucleotide polymorphism, SNP) in the von Willebrand factor gene detection that utilizes oligonucleotide arrays (DNA chips) on glass supports. Expoloring the association of the RsaI and SmaI polymorphisms in the von Willebrand factor gene with thrombosis in Chinese. Methods Allele specific oligonucleotides are covalently immobilized on glutaradehyde derivatized glass slides in arrays. Single strand PCR product of PCR amplified genomic DNA is fluorescently labeled by assymetric PCR with fluorescently tagged dUTP and hybridized to the support bound oligonucleotide array.The hybridization pattern is detected by fluorescence scenning. The effect of hybridization conditions were evaluated and optimized.The method was validated by the discrimination of blinded DNA samples identified by restriction enzymes. 50 patients with thrombosis were examined by this method. Results The genotypes scored by oligonucleotide arrays assay were in 100% agreement with restriction enzymes results. The genotype distributions GG, GA, AA and CC, CT, TT of RsaI and SmaI polymorphisms of patients or controls were 4.0%?12.0%?84 0% and 24 0%?44 0%?32.0% or 1.4%?11.8%?86.8% and 8.8%?57.4%?33 8%. The allele frequencies G, A and C,T were 10.0%,90.0% and 46.0%?54.0% or 7.4%?92.6% and 37 5%?62.5%. No statistical difference in genotype distribution and allele frequencies was observed between patients with thrombosis and the control groups (all P >0.05). Conclusion A rapid and accurate method for the RsaI and SmaI polymorphisms in the von Willebrand factor gene detection,or for the analysis of single nucleotide polymorphism (SNP) has been developed using oligonucleotide arrays on glass supports. No association was observed between the Rsa Ⅰ and Sma Ⅰ polymorphisms in the vWF gene and thrombotic disease.

关 键 词：寡核苷酸探针 多态性 血管性血友病 血栓性疾病 

分 类 号：R346[医药卫生—基础医学]