Single-nucleotide polymorphism (SNP) genotyping using cationic conjugated polymers in homogeneous solution
Single-nucleotide polymorphism (SNP) genotyping using cationic conjugated polymers in homogeneous solution
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DOI：
10.1038/nprot.2009.70
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发表时间：
2009-01-01
期刊：
影响因子：
14.8
通讯作者：
Wang, Shu
中科院分区：
文献类型：
--
作者：
Duan, Xinrui;Yue, Wei;Wang, Shu
该方案描述了一种简单、方便、灵敏的单核苷酸多态性（SNP）基因分型方法，该方法采用光学放大阳离子共轭聚合物和单碱基引物延伸反应。共轭聚合物（PFP，聚{（1,4-苯基）-2,7-[9,9-双（6'-N， N， N-三甲基铵）-己基芴]二溴}）与荧光素标记的dNTP （dNTP- fl）之间的荧光共振能量转移（FRET）效率与dNTP- fl加入等位基因特异性引物有关；当目标DNA和引物在SNP位点互补时，通过单碱基延伸反应合并。通过触发从PFP到荧光素的FRET，测量样品荧光强度的变化，可以区分SNP基因型。与其他SNP基因分型方法相比，该方案通过消除引物标记、繁琐的检查、化学/酶偶联反应和复杂的仪器，简化了程序并提高了灵敏度。PCR扩增大约需要2小时，然后5.5-7.5小时获得基因型。
This protocol describes a simple, convenient and sensitive single-nucleotide polymorphism (SNP) genotyping method using an optically amplifying cationic conjugated polymer and single base primer extension reaction. The fluorescence resonance energy transfer (FRET) efficiency between the conjugated polymer (PFP, poly{(1,4-phenylene)-2,7-[9,9-bis(6'-N, N, N-trimethyl ammonium)-hexyl fluorene] dibromide}) and a fluorescein-labeled dNTP (dNTP-Fl) is correlated to the incorporation of the dNTP-Fl into an allele-specific primer; incorporation occurs by a single base extension reaction when the target DNA and the primer are complementary at the SNP site. By triggering the FRET from PFP to fluorescein and measuring the change in fluorescence intensity of samples, the SNP genotypes can be discriminated. In comparison with other SNP genotyping methods, this protocol simplifies procedures and improves sensitivity by eliminating the need for primer labeling, cumbersome workups, chemical/enzymatic coupling reactions and sophisticated instruments. The assay takes about 2 h for PCR amplification followed by 5.5-7.5 h to obtain the genotypes.