Morinishi Leanna S, Blainey Paul
Broad Institute.
Department of Biological Engineering, Massachusetts Institute of Technology;
J Vis Exp. 2015 Sep 24(103):52925. doi: 10.3791/52925.
Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are still not routinely applied, due to the cost and specific equipment associated with commercially available methods. Here we present a simplified method for readout of digital droplet assays using a conventional real-time PCR instrument to measure bulk fluorescence of droplet-based digital assays. We characterize the performance of the bulk readout assay using synthetic droplet mixtures and a droplet digital multiple displacement amplification (MDA) assay. Quantitative MDA particularly benefits from a digital reaction format, but our new method applies to any digital assay. For established digital assay protocols such as digital PCR, this method serves to speed up and simplify assay readout. Our bulk readout methodology brings the advantages of partitioned assays without the need for specialized readout instrumentation. The principal limitations of the bulk readout methodology are reduced dynamic range compared with droplet-counting platforms and the need for a standard sample, although the requirements for this standard are less demanding than for a conventional real-time experiment. Quantitative whole genome amplification (WGA) is used to test for contaminants in WGA reactions and is the most sensitive way to detect the presence of DNA fragments with unknown sequences, giving the method great promise in diverse application areas including pharmaceutical quality control and astrobiology.
数字分析是一种强大的方法，能够检测稀有细胞并对单个核酸分子进行计数。然而，由于与市售方法相关的成本和特定设备，数字分析仍未得到常规应用。在此，我们提出一种简化方法，使用传统实时PCR仪器读取数字液滴分析的结果，以测量基于液滴的数字分析的总体荧光。我们使用合成液滴混合物和液滴数字多重置换扩增（MDA）分析来表征总体读数分析的性能。定量MDA尤其受益于数字反应形式，但我们的新方法适用于任何数字分析。对于诸如数字PCR等既定的数字分析方案，此方法有助于加快和简化分析读数。我们的总体读数方法带来了分区分析的优势，而无需专门的读数仪器。总体读数方法的主要局限性在于，与液滴计数平台相比，动态范围有所降低，并且需要标准样品，尽管对该标准的要求比对传统实时实验的要求低。定量全基因组扩增（WGA）用于测试WGA反应中的污染物，是检测未知序列DNA片段存在的最灵敏方法，这使得该方法在包括药物质量控制和天体生物学在内的各种应用领域具有广阔前景。
