Evaluating Digital PCR for the Quantification of Human Genomic DNA: Accessible Amplifiable Targets.
Evaluating Digital PCR for the Quantification of Human Genomic DNA: Accessible Amplifiable Targets.
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评估数字PCR的人类基因组DNA的定量：可访问的放大靶标。
DOI：
10.1021/acs.analchem.5b03692
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发表时间：
2016-02-16
影响因子：
7.4
通讯作者：
Duewer, David L.
中科院分区：
文献类型：
--
作者：
Kline, Margaret C.;Romsos, Erica L.;Duewer, David L.
当模板 DNA 的输入量控制在大约 √2 倍以内时，聚合酶链式反应 (PCR) 多重检测效果最佳。为了帮助确保 PCR 检测随时间和地点产生一致的结果，用于确定 DNA 数量的方法的结果需要在计量上可追溯到共同的参考。许多 DNA 定量系统可以使用 DNA 缓冲液溶液进行精确校准。由于不需要外部校准，终点有限稀释技术（统称为“数字 PCR (dPCR)”）已被提议适合对此类 DNA 校准物进行赋值。最近使用质粒、病毒或片段化基因组 DNA 记录了几种市售 dPCR 系统的性能特征；对更复杂材料（例如人类基因组 DNA）的 dPCR 性能研究较少。为了提供质量浓度可追溯认证的人类基因组参考材料，我们正在研究几种 dPCR 系统的测量特性。我们在这里报告了使用腔室和液滴 dPCR 平台对用四种核酸内切酶处理的四种人类基因组 DNA 进行多次 PCR 测定的测量结果。我们得出的结论是，dPCR 并不估计给定体积中 PCR 靶标的绝对数量，而是估计可访问和可扩增靶标的数量。虽然人类基因组 DNA 的酶促限制增加了某些检测的可及性，但在优化良好的 PCR 检测中，它可以减少可扩增靶标的数量，并增加相对于未切割样品的检测变异性。
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.
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影响因子：
4.3
作者：
Kline, Margaret C.;Duewer, David L.;Butler, John M.
通讯作者：
Butler, John M.
影响因子：
9.3
作者：
Huggett, Jim F.;Foy, Carole A.;Bustin, Stephen A.
通讯作者：
Bustin, Stephen A.
DOI：
10.1073/pnas.96.16.9236
发表时间：
1999-08-03
影响因子：
11.1
作者：
Vogelstein, B;Kinzler, KW
通讯作者：
Kinzler, KW
影响因子：
4.3
作者：
Duewer, David L.;Kline, Margaret C.;Romsos, Erica L.
通讯作者：
Romsos, Erica L.
影响因子：
7.4
作者：
Kline, MC;Duewer, DL;Butler, JM
通讯作者：
Butler, JM