Production and certification of NIST Standard Reference Material 2372 Human DNA Quantitation Standard
Production and certification of NIST Standard Reference Material 2372 Human DNA Quantitation Standard
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DOI：
10.1007/s00216-009-2782-0
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发表时间：
2009-06-01
影响因子：
4.3
通讯作者：
Butler, John M.
中科院分区：
文献类型：
--
作者：
Kline, Margaret C.;Duewer, David L.;Butler, John M.
法医人类身份鉴定界使用的现代高度多重短串联重复序列（STR）检测需要严格控制聚合酶链反应（PCR）过程中扩增的样本DNA的初始量。这反过来又需要能够重复测量样品提取物中人类DNA [DNA]的浓度。定量PCR（qPCR）技术可以在极小的样品中确定特定核苷酸序列的完整DNA片段的数量;然而，这些测定必须使用具有良好表征和稳定组成的DNA提取物进行校准。到2004年，由美国国家标准与技术研究所（NIST）协调或向其报告的研究表明，一种表征良好、稳定的人类DNA定量认证参考物质（CRM）可以帮助法医界减少实验室内和实验室外的定量变异性。为了确保可以监测这种定量标准品的稳定性，并且在需要时可以制备等效的替代材料，需要测量与[DNA]直接相关的一些稳定量。使用260 nm处的光密度（正确地称为十进制衰减）与水溶液中的[DNA]之间长期建立的传统关系，NIST标准参考物质（SRM）2372人类DNA定量标准品于2007年10月发布。这个SRM由三个完全不同的DNA提取物组成：一个单源男性，一个多源女性，以及男性和女性来源的混合物。所有三种SRM组分具有非常相似的光密度，因此非常相似的常规[DNA]。这些材料在几种广泛使用的性别中性测定中的表现非常相似，表明适当的制备方法和计量学上合理的分光光度测量的组合能够制备和认证定量[DNA]标准品，这些标准品既可维护又具有实用性。
Modern highly multiplexed short tandem repeat (STR) assays used by the forensic human-identity community require tight control of the initial amount of sample DNA amplified in the polymerase chain reaction (PCR) process. This, in turn, requires the ability to reproducibly measure the concentration of human DNA, [DNA], in a sample extract. Quantitative PCR (qPCR) techniques can determine the number of intact stretches of DNA of specified nucleotide sequence in an extremely small sample; however, these assays must be calibrated with DNA extracts of well-characterized and stable composition. By 2004, studies coordinated by or reported to the National Institute of Standards and Technology (NIST) indicated that a well-characterized, stable human DNA quantitation certified reference material (CRM) could help the forensic community reduce within-and among-laboratory quantitation variability. To ensure that the stability of such a quantitation standard can be monitored and that, if and when required, equivalent replacement materials can be prepared, a measurement of some stable quantity directly related to [DNA] is required. Using a long-established conventional relationship linking optical density (properly designated as decadic attenuance) at 260 nm with [DNA] in aqueous solution, NIST Standard Reference Material (SRM) 2372 Human DNA Quantitation Standard was issued in October 2007. This SRM consists of three quite different DNA extracts: a single-source male, a multiple-source female, and a mixture of male and female sources. All three SRM components have very similar optical densities, and thus very similar conventional [DNA]. The materials perform very similarly in several widely used gender-neutral assays, demonstrating that the combination of appropriate preparation methods and metrologically sound spectrophotometric measurements enables the preparation and certification of quantitation [DNA] standards that are both maintainable and of practical utility.