用于单核苷酸多态性基因分型的化学梯度介导熔解曲线分析
Chemical gradient-mediated melting curve analysis for genotyping of SNPs.
作者信息
Russom Aman, Irimia Daniel, Toner Mehmet
机构信息
Center for Engineering in Medicine, Massachusetts General Hospital, Harvard Medical School and Shriners Hospital for Children, Boston, MA 02114, USA.
出版信息
Electrophoresis. 2009 Jul;30(14):2536-43. doi: 10.1002/elps.200800729.
This report describes a microfluidic solid-phase chemical gradient-mediated melting curve analysis method for SNP analysis. The method is based on allele-specific denaturation to discriminate mismatched (MM) from perfectly matched (PM) DNA duplexes upon exposure to linear chemical gradient. PM and MM DNA duplexes conjugated on beads are captured in a microfluidic gradient generator device designed with dams, keeping the beads trapped perpendicular to a gradient generating channel. Two denaturants, formamide and urea, were tested for their ability to destabilize the DNA duplex by competing with Watson-Crick pairing. Upon exposure to the chemical gradient, rapid denaturing profile was monitored in real time using fluorescence microscopy. The results show that the two duplexes exhibit different kinetics of denaturation profiles, enabling discrimination of MM from PM DNA duplexes to score SNP.
本报告描述了一种用于单核苷酸多态性（SNP）分析的微流控固相化学梯度介导的熔解曲线分析方法。该方法基于等位基因特异性变性，在暴露于线性化学梯度时区分错配（MM）和完全匹配（PM）的DNA双链体。结合在珠子上的PM和MM DNA双链体被捕获在一个设计有坝的微流控梯度发生器装置中，使珠子垂直于梯度产生通道被困住。测试了两种变性剂甲酰胺和尿素通过与沃森-克里克配对竞争来破坏DNA双链体稳定性的能力。在暴露于化学梯度时，使用荧光显微镜实时监测快速变性曲线。结果表明，这两种双链体表现出不同的变性曲线动力学，能够区分MM和PM DNA双链体以对SNP进行评分。
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