Decraene Charles, Bortolini Silveira Amanda, Michel Marc, Bidard François-Clément, Pierga Jean-Yves, Stern Marc-Henri, Proudhon Charlotte
Circulating Tumor Biomarkers Laboratory, SiRIC, Translational Research Department, Institut Curie, PSL Research University; CNRS UMR144, Institut Curie, PSL Research University.
Circulating Tumor Biomarkers Laboratory, SiRIC, Translational Research Department, Institut Curie, PSL Research University.
J Vis Exp. 2018 Sep 25(139):58051. doi: 10.3791/58051.
Droplet digital polymerase chain reaction (ddPCR) is a highly sensitive quantitative polymerase chain reaction (PCR) method based on sample fractionation into thousands of nano-sized water-in-oil individual reactions. Recently, ddPCR has become one of the most accurate and sensitive tools for circulating tumor DNA (ctDNA) detection. One of the major limitations of the standard ddPCR technique is the restricted number of mutations that can be screened per reaction, as specific hydrolysis probes recognizing each possible allelic version are required. An alternative methodology, the drop-off ddPCR, increases throughput, since it requires only a single pair of probes to detect and quantify potentially all genetic alterations in the targeted region. Drop-off ddPCR displays comparable sensitivity to conventional ddPCR assays with the advantage of detecting a greater number of mutations in a single reaction. It is cost-effective, conserves precious sample material, and can also be used as a discovery tool when mutations are not known a priori.
液滴数字聚合酶链反应（ddPCR）是一种高度灵敏的定量聚合酶链反应（PCR）方法，它基于将样本分成数千个纳米级的油包水单个反应。最近，ddPCR已成为循环肿瘤DNA（ctDNA）检测最准确、最灵敏的工具之一。标准ddPCR技术的主要局限性之一是每个反应可筛查的突变数量有限，因为需要识别每个可能等位基因版本的特异性水解探针。另一种方法，即脱落式ddPCR，提高了通量，因为它仅需一对探针就能检测和定量目标区域中潜在的所有基因改变。脱落式ddPCR与传统ddPCR检测方法具有相当的灵敏度，其优势在于能在单个反应中检测到更多的突变。它具有成本效益，能节省珍贵的样本材料，并且当事先不知道突变情况时，还可作为一种发现工具使用。
