Technology Development Unit, Omics Science Center (OSC), RIKEN Yokohama Institute, Kanagawa, Japan.
Hum Mutat. 2010 Feb;31(2):208-17. doi: 10.1002/humu.21177.
Most commonly used intercalating fluorescent dyes in DNA detection are lacking any sequence specificity, whereas so-called Exciton Primers can overcome this limitation by functioning as "sequence-specific dyes." After hybridization to complementary sequences, the fluorescence of Exciton Primers provides sequence-specific signals for real-time monitoring of amplification reactions. Applied to the SmartAmp2 mutation detection process, Exciton Primers show high signal strength with low background leading to a superior specificity and sensitivity compared to SYBR Green I. Signal strength can be further enhanced using multiple dyes within one Exciton Primer or use of multiple Exciton Primers in the same amplification reaction. Here we demonstrate the use of Exciton Primers for genotyping a single nucleotide polymorphism (SNP) in the VKORC1 locus (-1639G>A) relevant for Warfarin dosing as an example for Exciton Primers mediated genotyping by SmartAmp2. The genotyping assay can use only one labeled Exciton Primer for endpoint detection, or simultaneously by real-time monitoring detect wild-type and mutant alleles in a one-tube reaction using two Exciton Primers having different dyes. Working directly from blood samples, Exciton Primer mediated genotyping by SmartAmp2 offers superior solutions for rapid point-of-care testing.
最常用于 DNA 检测的荧光染料缺乏任何序列特异性，而所谓的激发子引物可以通过作为“序列特异性染料”来克服这一限制。在与互补序列杂交后，激发子引物的荧光为实时监测扩增反应提供了序列特异性信号。将激发子引物应用于 SmartAmp2 突变检测过程中，与 SYBR Green I 相比，其具有低背景的高信号强度，从而具有更高的特异性和灵敏度。通过在一个激发子引物中使用多种染料或在同一扩增反应中使用多个激发子引物，可以进一步增强信号强度。在这里，我们以 VKORC1 基因座（-1639G>A）中与华法林剂量相关的单核苷酸多态性（SNP）基因为例，展示了激发子引物在 SmartAmp2 介导的基因分型中的应用。该基因分型检测可以仅使用一个标记的激发子引物进行终点检测，或者在一个管中同时通过实时监测，使用具有不同染料的两个激发子引物检测野生型和突变等位基因。直接从血液样本中进行，SmartAmp2 介导的激发子引物基因分型提供了用于快速即时护理测试的卓越解决方案。
