Digital Assays Part I: Partitioning Statistics and Digital PCR
Digital Assays Part I: Partitioning Statistics and Digital PCR
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DOI：
10.1177/2472630317705680
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发表时间：
2017-08-01
期刊：
影响因子：
2.7
通讯作者：
Basu, Amar S.
中科院分区：
文献类型：
--
作者：
Basu, Amar S.
数字测定是其中样品被划分到许多小容器中使得每个分区包含离散数量的生物实体（0、1、2、3、.）的测定。作为生物学家工具包中的一项强大技术，数字分析在定量核酸、测量蛋白质及其酶活性以及探测单细胞基因型和表型方面带来了新的精确度。本文的第一部分从分区的好处和泊松统计开始，包括错误的来源。其余的重点是用于核酸定量的数字PCR（dPCR）。我们讨论了五个商业仪器，分区样品到物理隔离室（cdPCR）或液滴乳液（ddPCR）。我们比较了dPCR的优势（绝对定量，精密度和检测稀有或突变目标的能力）与其前身定量实时PCR（动态范围，更大的样品体积和吞吐量）。最后，我们描述了dPCR的几个有前途的应用，包括拷贝数变异，定量循环肿瘤DNA和病毒载量，RNA/miRNA定量与逆转录dPCR，和下一代测序的文库制备。这篇综述旨在为有兴趣将数字分析纳入其工作流程的科学家提供广泛的视角。第二部分重点介绍数字蛋白质和细胞分析。
A digital assay is one in which the sample is partitioned into many small containers such that each partition contains a discrete number of biological entities (0, 1, 2, 3, ...). A powerful technique in the biologist's toolkit, digital assays bring a new level of precision in quantifying nucleic acids, measuring proteins and their enzymatic activity, and probing single-cell genotypes and phenotypes. Part I of this review begins with the benefits and Poisson statistics of partitioning, including sources of error. The remainder focuses on digital PCR (dPCR) for quantification of nucleic acids. We discuss five commercial instruments that partition samples into physically isolated chambers (cdPCR) or droplet emulsions (ddPCR). We compare the strengths of dPCR (absolute quantitation, precision, and ability to detect rare or mutant targets) with those of its predecessor, quantitative real-time PCR (dynamic range, larger sample volumes, and throughput). Lastly, we describe several promising applications of dPCR, including copy number variation, quantitation of circulating tumor DNA and viral load, RNA/miRNA quantitation with reverse transcription dPCR, and library preparation for next-generation sequencing. This review is intended to give a broad perspective to scientists interested in adopting digital assays into their workflows. Part II focuses on digital protein and cell assays.