Bristol Genetic Epidemiology Laboratory and MRC Centre for Causal Analyses in Translational Epidemiology, School of Social and Community Medicine, University of Bristol, Oakfield Grove, Clifton BS8 2BN, UK.
Nucleic Acids Res. 2011 Apr;39(8):e54. doi: 10.1093/nar/gkr046. Epub 2011 Feb 7.
We describe a generic design for ratiometric analysis suitable for determination of copy number variation (CNV) class of a gene. Following two initial sequence-specific PCR priming cycles, both ends of both amplicons (one test and one reference) in a duplex reaction, are all primed by the same universal primer (UP). Following each amplification denaturation step, the UP target and its reverse complement (UP') in each strand form a hairpin. The bases immediately beyond the 3'-end of the UP and 5' of UP' are chosen such as not to base pair in the hairpin (otherwise priming is ablated). This hairpin creates a single constant environment for priming events and chaperones free 3'-ends of amplicon strands. The resultant 'amplification ratio control system' (ARCS) permits ratiometric representation of amplicons relative to the original template into PCR plateau phase. These advantages circumvent the need for real-time PCR for quantitation. Choice of different %(G+C) content for the target and reference amplicons allows liquid phase thermal melt discrimination and quantitation of amplicons. The design is generic, simple to set up and economical. Comparisons with real-time PCR and other techniques are made and CNV assays demonstrated for haptoglobin duplicon and 'chemokine (C-C motif) ligand 3-like 1' gene.
我们描述了一种适用于基因拷贝数变异 (CNV) 类别的比率分析的通用设计。在双反应中，经过两轮初始的序列特异性 PCR 引物循环后，两个扩增子（一个测试和一个参考）的两端都由同一个通用引物 (UP) 进行引物延伸。在每次扩增变性步骤后，每个链上的 UP 靶标及其反向互补序列 (UP') 形成发夹结构。选择 UP 的 3'末端和 UP'的 5'端的碱基，使其不在发夹结构中配对（否则引物延伸就会被破坏）。这个发夹结构为引物延伸事件创造了一个单一的恒定环境，并为扩增子链的 3'末端提供了无伴侣的自由。由此产生的“扩增比率控制系统” (ARCS) 允许在 PCR 平台相阶段，对相对于原始模板的扩增子进行比率表示。这些优势避免了实时 PCR 定量的需要。选择目标和参考扩增子的不同 %(G+C) 含量允许进行液相热融解区分和扩增子的定量。该设计具有通用性，易于设置，且经济实惠。我们对其与实时 PCR 和其他技术进行了比较，并对结合珠蛋白重复序列和“趋化因子 (C-C 基元) 配体 3 样 1”基因进行了 CNV 检测。
