Evaluating Digital PCR for the Quantification of Human Genomic DNA: Accessible Amplifiable Targets

Polymerase chain reaction (PCR) multiplexed assays perform best when the input quantity of template DNA is controlled to within about a factor of √2. To help ensure that PCR assays yield consistent results over time and place, results from methods used to determine DNA quantity need to be metrologically traceable to a common reference. Many DNA quantitation systems can be accurately calibrated with solutions of DNA in aqueous buffer. Since they do not require external calibration, end-point limiting dilution technologies, collectively termed “digital PCR (dPCR)”, have been proposed as suitable for value assigning such DNA calibrants. The performance characteristics of several commercially available dPCR systems have recently been documented using plasmid, viral, or fragmented genomic DNA; dPCR performance with more complex materials, such as human genomic DNA, has been less studied. With the goal of providing a human genomic reference material traceably certified for mass concentration, we are investigating the measurement characteristics of several dPCR systems. We here report results of measurements from multiple PCR assays, on four human genomic DNAs treated with four endonuclease restriction enzymes using both chamber and droplet dPCR platforms. We conclude that dPCR does not estimate the absolute number of PCR targets in a given volume but rather the number of accessible and amplifiable targets. While enzymatic restriction of human genomic DNA increases accessibility for some assays, in well-optimized PCR assays it can reduce the number of amplifiable targets and increase assay variability relative to uncut sample.

聚合酶链式反应（Polymerase chain reaction, PCR）多重检测的性能最优条件为，将模板DNA的输入量控制在约√2倍的范围内。为确保PCR检测在不同时间与地点均能获得一致结果，用于DNA定量的检测方法所得结果需具备计量可溯源性，可溯源至统一参考标准。多数DNA定量系统可通过水溶液缓冲液中的DNA溶液实现精准校准。由于无需外部校准，被统称为“数字PCR（digital PCR, dPCR）”的终点极限稀释技术已被证实适用于此类DNA校准品的赋值工作。近期已有研究采用质粒、病毒或片段化基因组DNA，对多款商用dPCR系统的性能特征进行了表征；但针对更复杂样本（如人类基因组DNA）的dPCR性能研究相对较少。为了提供经质量浓度溯源认证的人类基因组参考物质，我们正对多款dPCR系统的测量特性展开研究。本文报道了多组PCR检测的结果：我们对四份人类基因组DNA样本分别采用四种限制性内切酶进行处理，并使用舱室式与微滴式两种dPCR平台开展检测。我们的研究结论表明：dPCR并非估算给定体积内PCR靶标的绝对数量，而是可接触且可扩增的靶标数量。尽管对人类基因组DNA进行酶切可提升部分检测的可接触性，但在优化良好的PCR检测中，与未酶切的样本相比，酶切处理反而可能降低可扩增靶标的数量，并加剧检测的变异程度。