Single‐nucleotide polymorphism analysis by allele‐specific extension of fluorescently labeled nucleotides in a microfluidic flow‐through device
Single‐nucleotide polymorphism analysis by allele‐specific extension of fluorescently labeled nucleotides in a microfluidic flow‐through device
复制标题
通过微流体流通装置中荧光标记核苷酸的等位基因特异性延伸进行单核苷酸多态性分析
DOI：
10.1002/elps.200390008
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发表时间：
2003
期刊：
影响因子：
2.9
通讯作者：
G. Stemme
中科院分区：
文献类型：
--
作者：
A. Russom;A. Ahmadian;H. Andersson;P. Nilsson;G. Stemme
我们描述了一种用于荧光标记核苷酸的等位基因特异性延伸的微流体方法，用于对单核苷酸多态性（SNP）进行评分。该方法利用了与 DNA 模板杂交的等位基因特异性引物的匹配和不匹配配置之间的反应动力学不同的事实。用于珠上生化反应的微流体流通装置被用来利用反应动力学来提高 DNA 聚合酶的序列特异性，从而区分不匹配的构型和匹配的构型。反应室的容积为12.5nL。使用我们的方法对 p53 基因密码子 72 处的 SNP 位点的所有三种可能变异进行评分。这项工作证明了通过微流体流通装置中荧光标记核苷酸的等位基因特异性延伸对 SNP 进行评分的可能性。微流控装置的灵敏检测系统和简单的微加工能够进一步小型化和生产用于高通量 SNP 分析的阵列格式的微流控装置。
We describe a microfluidic approach for allele‐specific extension of fluorescently labeled nucleotides for scoring of single‐nucleotide polymorphism (SNP). The method takes advantage of the fact that the reaction kinetics differs between matched and mismatched configurations of allele‐specific primers hybridized to DNA template. A microfluidic flow‐through device for biochemical reactions on beads was used to take advantage of the reaction kinetics to increase the sequence specificity of the DNA polymerase, discriminating mismatched configurations from matched. The volume of the reaction chamber was 12.5 nL. All three possible variants of an SNP site at codon 72 of the p53 gene were scored using our approach. This work demonstrates the possibility of scoring SNP by allele‐specific extension of fluorescently labeled nucleotides in a microfluidic flow‐through device. The sensitive detection system and easy microfabrication of the microfluidic device enable further miniaturization and production of an array format of microfluidic devices for high‐throughput SNP analysis.