Institute of Legal Medicine, University of Münster, Röntgenstr. 23, 48149, Münster, Germany.
Institute of Legal Medicine, University of Ulm, Albert-Einstein-Allee 23, 89081, Ulm, Germany.
Forensic Sci Med Pathol. 2023 Mar;19(1):50-59. doi: 10.1007/s12024-022-00549-3. Epub 2022 Nov 10.
Several commercially available quantitative real-time PCR (qPCR) systems enable highly sensitive detection of human DNA and provide a degradation index (DI) to assess DNA quality. From routine casework in forensic genetics, it was observed that DNA degradation in forensic samples such as blood samples stored under sub-optimal conditions leads to visible effects in multiplex analyses of short tandem repeat markers (STRs) due to decreased amplification efficiencies in longer amplicons. It was further noticed that degradation indices often remain below the value that is considered to be critical. Thus, the aim of this work was to systematically analyze this effect and to compare conventional qPCR assays with a modified qPCR approach using uracil DNA glycosylase (UNG) and DNA quality assessment methods based on electrophoresis. Blood samples were stored at three different storage temperatures for up to 316 days. Significantly increased DNA recovery was observed from samples stored at high temperatures (37 °C) compared samples stored at room temperature and 4 °C. We observed typical effects of degradation in STR analyses but no correlation between DI and storage time in any of the storage conditions. Adding UNG slightly increased the sensitivity of detecting DNA degradation in one of the qPCR kits used in this study. This observation was not confirmed when using a second qPCR system. Electrophoretic systems did also not reveal significant correlations between integrity values and time. Methods for detecting DNA degradation are usually limited to the detection of DNA fragmentation, and we conclude that degradation affecting forensic STR typing is more complex.
几种市售的定量实时 PCR（qPCR）系统能够高度灵敏地检测人 DNA，并提供降解指数（DI）来评估 DNA 质量。从法医遗传学的常规案例工作中观察到，在储存条件不理想的情况下，法医样本（如血液样本）中的 DNA 降解会导致短串联重复标记（STR）的多重分析中出现可见的影响，因为较长的扩增子的扩增效率降低。进一步注意到，降解指数往往仍低于被认为是关键的数值。因此，这项工作的目的是系统地分析这种影响，并将常规 qPCR 检测与使用尿嘧啶 DNA 糖基化酶（UNG）的改良 qPCR 方法以及基于电泳的 DNA 质量评估方法进行比较。血液样本在三种不同的储存温度下储存长达 316 天。与在室温下和 4°C 下储存的样本相比，在高温（37°C）下储存的样本中观察到明显增加的 DNA 回收。我们观察到 STR 分析中典型的降解效应，但在任何储存条件下，DI 与储存时间之间均无相关性。在本研究中使用的一种 qPCR 试剂盒中添加 UNG 略微提高了检测 DNA 降解的灵敏度。当使用第二种 qPCR 系统时，并未证实这一观察结果。电泳系统也未显示完整性值与时间之间存在显著相关性。用于检测 DNA 降解的方法通常仅限于检测 DNA 片段化，我们得出结论，影响法医 STR 分型的降解更为复杂。
