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基于焦磷酸测序技术建立阿司匹林个体化用药相关SNP位点的分型方法
投稿时间:2017-04-27  修订日期:2017-05-09  点此下载全文
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作者单位E-mail
邢晓清 河北省人民医院 zkymw2013@163.com 
卜修民 中国药科大学  
宋沁馨 中国药科大学 songqinxin@cpu.edu.cn 
基金项目:国家自然科学基金面上项目(No. 81673390);江苏省重点研发计划(社会发展)项目(No. BE2016745);江苏省基础研究计划(自然科学基金)项目(No. BK20151445);药物质量与安全预警教育部重点实验室资助项目(No. DQCP2015MS02);中国药科大学药学基地科研训练及科研能力提高项目(No. J1310032);江苏省青蓝工程资助
中文摘要:阿司匹林的抗血小板作用在临床存在显著的个体差异,而rs5918、rs12041331和rs730012三个单核苷酸多态性位点与阿司匹林的药效和副作用有着密切的联系。本文基于PCR结合焦磷酸测序技术,建立了阿司匹林相关SNP位点的基因分型方法。自主设计扩增引物和测序引物;优化PCR扩增条件,使3个位点能够在相同的条件下扩增并通过梯度稀释基因组DNA检测方法的灵敏度;最后,分别基于本文建立的新方法和Sanger测序技术对20例临床样本进行三个位点的基因分型,验证该方法的准确性。结果显示,本文成功建立了阿司匹林个体化用药相关SNP位点的基因分型方法,检测下限低至0.4 ng 基因组DNA,用两种方法对20例临床样本进行三个位点的基因分型,结果完全一致,说明该方法有较高的准确性。
中文关键词:阿司匹林  焦磷酸测序技术  单核苷酸多态性  个体化用药
 
Establishment of Genotype Method for SNPs related to Individualized Aspirin Treatment Based on Pyrosequencing Technology
Abstract:There are significant individual differences in the antiplatelet effects of aspirin, three single nucleotide polymorphisms (SNPs) are reported to significantly correlated with the efficacy and side effects of aspirin:rs5918, rs12041331 and rs730012. The present study established the genotyping method of the three SNPs based on the combination of polymerase chain reaction and pyrosequencing technology. Amplification primers and sequencing primers were designed independently, the amplification conditions were optimized to make the three SNPs amplified in the same condition. The sensitivity of the method was detected by using starting genome DNA in different concentration. In order to testify the accuracy of the method, the proposed method and Sanger sequencing technology were both used for the three SNPs genotyping in 20 blood samples. The results illustrated that we successfully established the genotype method of aspirin-related SNPs, the detection limit as low as 0.4 ng genome DNA. The genotype results of 20 samples by the proposed method were exactly same as that of sanger sequencing. It is evident that the proposed method is sensitive and accurate.
keywords:Aspirin  Pyrosequencing  Single nucleotide polymorphism  Personalized medicine
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