National Measurement Institute, Lindfield, New South Wales, Australia.
Anal Chem. 2010 Sep 1;82(17):7185-92. doi: 10.1021/ac100845m.
Accurate estimation of total DNA concentration (mass concentration, e.g., ng/muL) that is traceable to the International System of Units (SI) is a crucial starting point for improving reproducible measurements in many applications involving nucleic acid testing and requires a DNA reference material which has been certified for its total DNA concentration. In this study, the concentrations of six different lambda DNA preparations were determined using different measurement platforms: UV Absorbance at 260 nm (A(260)) with and without prior sodium hydroxide (NaOH) treatment of the DNA, PicoGreen assay, and digital polymerase chain reaction (dPCR). DNA concentration estimates by A(260) with and without prior NaOH treatment were significantly different for five of the six samples tested. There were no significant differences in concentration estimates based on A(260) with prior NaOH treatment, PicoGreen analysis, and dPCR for two of the three samples tested using dPCR. Since the measurand in dPCR is amount (copy number) concentration (copies/muL), the results suggest that accurate estimation of DNA mass concentration based on copy number concentration is achievable provided the DNA is fully characterized and in the double-stranded form or amplification is designed to be initiated from only one of the two complementary strands.
准确估计总 DNA 浓度（质量浓度，例如 ng/μL），并溯源至国际单位制（SI），是许多涉及核酸检测的应用中提高可重复性测量的关键起点，这需要一种总 DNA 浓度经过认证的 DNA 参考物质。在本研究中，使用不同的测量平台（260nm 处的紫外吸光度（A(260)），有无 DNA 先用氢氧化钠（NaOH）处理，PicoGreen 分析和数字聚合酶链反应（dPCR）），测定了六种不同 lambda DNA 制剂的浓度。对于测试的六种样本中的五种，未经 NaOH 处理的 A(260)和经 NaOH 处理的 A(260)的 DNA 浓度估计值存在显著差异。对于使用 dPCR 测试的三个样本中的两个，经 NaOH 处理的 A(260)、PicoGreen 分析和 dPCR 的浓度估计值没有显著差异。由于 dPCR 中的可测量值是量（拷贝数）浓度（拷贝数/μL），因此这些结果表明，只要 DNA 完全表征为双链形式或设计仅从两条互补链之一起始扩增，基于拷贝数浓度准确估计 DNA 质量浓度是可行的。
