Zhang Jinpei, Li Lu, Bai Xue, Zhang Zhe, Yuan Li
Engineering Research Center of Crime Scene Evidence Examination, Beijing, 100038, PR China.
Collaborative Innovation Center of Judicial Civilization, Beijing, 100088, PR China.
Int J Legal Med. 2025 May;139(3):995-1003. doi: 10.1007/s00414-024-03391-0. Epub 2024 Dec 10.
Formalin-fixed tissues possess irreplaceable value as a source of DNA for identification, especially when fresh samples are unavailable. Nonetheless, extracting and amplifying DNA from these tissues is challenging, primarily due to formaldehyde-induced cross-linking and nucleic acid fragmentation. In this study, two pre-extraction treatments, gradual dehydration using ethanol and pre-digestion heat treatments, and three DNA extraction methods, the Chelex-100 method, TIANamp FFPE DNA Kit, and ML Ultra-micro DNA extraction kit, were utilized to optimize DNA extraction from different tissues, which were fixed in 4% unbuffered formalin for different durations. The tissues include the heart, liver, spleen, lung, kidney, muscle, and brain. DNA quality was assessed, and quantification was conducted using Spectrophotometer and Quantifiler Trio DNA Quantification Kits, while the GSTAR™ 25 kit was employed for STR detection. The results indicated that the two pre-extraction treatments exhibited no significant effect on the STR success rate. On day 9, allelic dropout was observed in the heart, liver, spleen, lung, and kidney tissues. Furthermore, allelic dropout was observed in muscle and brain at 12 days and 15 days, respectively. In conclusion, the results underscore the feasibility of effectively extracting DNA from formalin-fixed tissues within 9 days for subsequent STR analysis.
福尔马林固定组织作为用于鉴定的DNA来源具有不可替代的价值，尤其是在无法获得新鲜样本时。尽管如此，从这些组织中提取和扩增DNA具有挑战性，主要是由于甲醛诱导的交联和核酸片段化。在本研究中，采用两种提取前处理方法，即乙醇梯度脱水和预消化热处理，以及三种DNA提取方法，即Chelex-100法、TIANamp FFPE DNA试剂盒和ML超微量DNA提取试剂盒，对固定于4%无缓冲福尔马林中不同时长的不同组织进行DNA提取优化。这些组织包括心脏、肝脏、脾脏、肺、肾脏、肌肉和大脑。使用分光光度计和Quantifiler Trio DNA定量试剂盒评估DNA质量并进行定量，同时使用GSTAR™ 25试剂盒进行STR检测。结果表明，两种提取前处理方法对STR成功率无显著影响。在第9天，在心脏、肝脏、脾脏、肺和肾脏组织中观察到等位基因缺失。此外，在第12天和第15天分别在肌肉和大脑中观察到等位基因缺失。总之，结果强调了在9天内从福尔马林固定组织中有效提取DNA用于后续STR分析的可行性。
