Single nucleotide polymorphism analysis by allele-specific primer extension with real-time bioluminescence detection in a microfluidic device
Single nucleotide polymorphism analysis by allele-specific primer extension with real-time bioluminescence detection in a microfluidic device
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DOI：
10.1016/s0021-9673(03)01033-1
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发表时间：
2003-10-03
影响因子：
4.1
通讯作者：
Stemme, G
中科院分区：
文献类型：
--
作者：
Russom, A;Tooke, N;Stemme, G
提出了一种快速生物发光实时检测单核苷酸多态性 (SNP) 的微流体方法。该方法基于使用焦磷酸测序化学的单步引物延伸来实时监测核苷酸掺入。该方法利用了匹配和不匹配的引物模板配置之间的反应动力学不同的事实。我们在此表明，通过比较匹配和不匹配配置之间的初始反应动力学，实时监控初始反应可以准确地对 SNP 进行评分。因此，不需要额外的处理来提高延伸的序列特异性，这对于许多等位基因特异性延伸测定来说都是如此。使用四个 SNP 评估微流体方法。其中三个 SNP 包含引物模板配置，此前已报道这些配置很难通过等位基因特异性引物延伸来解析。所有调查的 SNP 均已成功评分。使用微流控装置，生物发光测定的体积显着减小，从而提供了一种经济有效且快速的SNP分析方法。 (C) 2003 Elsevier B.V. 保留所有权利。
A microfluidic approach for rapid bioluminescent real-time detection of single nucleotide polymorphism (SNP) is presented. The method is based on single-step primer extension using pyrosequencing chemistry to monitor nucleotide incorporations in real-time. The method takes advantage of the fact that the reaction kinetics differ between matched and mismatched primer-template configurations. We show here that monitoring the initial reaction in real time accurately scores SNPs by comparing the initial reaction kinetics between matched and mismatched configurations. Thus, no additional treatment is required to improve the sequence specificity of the extension, which has been the case for many allele-specific extension assays. The microfluidic approach was evaluated using four SNPs. Three of the SNPs included primer-template configurations that have been previously reported to be difficult to resolve by allele-specific primer extension. All SNPs investigated were successfully scored. Using the microfluidic device, the volume for the bioluminescent assay was reduced dramatically, thus offering a cost-effective and fast SNP analysis method. (C) 2003 Elsevier B.V. All rights reserved.