Enhanced Ratio of Signals Enables Digital Mutation Scanning for Rare Allele Detection
Enhanced Ratio of Signals Enables Digital Mutation Scanning for Rare Allele Detection
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DOI：
10.1016/j.jmoldx.2014.12.003
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发表时间：
2015-05-01
影响因子：
4.1
通讯作者：
Makrigiorgos, G. Mike
中科院分区：
文献类型：
--
作者：
Castellanos-Rizaldos, Elena;Paweletz, Cloud;Makrigiorgos, G. Mike
微滴数字PCR（ddPCR）用于癌症、产前诊断和传染病中的低水平DNA突变检测的应用正在迅速增长。然而，尽管ddPCR已经成功地用于检测预定位置处的罕见突变，但是不存在用于突变扫描的ddPCR适应。然而，临床相关突变常常位于肿瘤抑制基因的多个序列位置或癌基因的复杂热点突变。在这里，我们描述了在较低的变性温度PCR（COLD-PCR）与ddPCR，使数字突变扫描内约50 bp的部分的目标扩增子的组合。在ddPCR过程中使用两种与野生型序列匹配的FAM/HEX标记的水解探针。当扩增野生型扩增子时，FAM/HEX阳性液滴的比率是恒定的，但当存在FAM或HEX探针下任何位置的突变时，FAM/HEX阳性液滴的比率偏离。为了增强FAM/HEX比率的变化，我们采用了COLD-PCR循环条件，其富集序列上任何地方的含突变扩增子。我们使用连续突变稀释液和无细胞循环DNA样本验证了TP 53和EGFR中多个突变的COLD-ddPCR，并证明检测到约0.2%至1.2%的突变丰度。COLD-ddPCR实现了一种简单、快速和稳健的双荧光检测方法，用于在ddPCR期间鉴定多个突变，并可能鉴定靶序列中存在的未知DNA变体。
The use of droplet digital PCR (ddPCR) for low-Level DNA mutation detection in cancer, prenatal diagnosis, and infectious diseases is growing rapidly. However, although ddPCR has been implemented successfully for detection of rare mutations at pre-determined positions, no ddPCR adaptation for mutation scanning exists. Yet, frequently, clinically relevant mutations reside on multiple sequence positions in tumor suppressor genes or complex hotspot mutations in oncogenes. Here, we describe a combination of coamplification at Lower denaturation temperature PCR (COLD-PCR) with ddPCR that enables digital mutation scanning within approximately 50-bp sections of a target amplicon. Two FAM/HEX-labeled hydrolysis probes matching the wild-type sequence are used during ddPCR. The ratio of FAM/HEX-positive droplets is constant when wild-type amplicons are amplified but deviates when mutations anywhere under the FAM or HEX probes are present. To enhance the change in FAM/HEX ratio, we employed COLD-PCR cycling conditions that enrich mutation-containing amplicons anywhere on the sequence. We validated COLD-ddPCR on multiple mutations in TP53 and in EGFR using serial mutation dilutions and cell-free circulating DNA samples, and demonstrate detection down to approximately 0.2% to 1.2% mutation abundance. COLD-ddPCR enables a simple, rapid, and robust two-fluorophore detection method for the identification of multiple mutations during ddPCR and potentially can identify unknown DNA variants present in the target sequence.