Royal Canadian Mounted Police, National Services and Research, 1200 Vanier Parkway, Ottawa, Ontario K1A 0R2, Canada.
Forensic Sci Int Genet. 2010 Feb;4(2):80-8. doi: 10.1016/j.fsigen.2009.06.003. Epub 2009 Jul 8.
Archival tissue preserved in fixative constitutes an invaluable resource for histological examination, molecular diagnostic procedures and for DNA typing analysis in forensic investigations. However, available material is often limited in size and quantity. Moreover, recovery of DNA is often severely compromised by the presence of covalent DNA-protein cross-links generated by formalin, the most prevalent fixative. We describe the evaluation of buffer formulations, sample lysis regimens and DNA recovery strategies and define optimized manual and automated procedures for the extraction of high quality DNA suitable for molecular diagnostics and genotyping. Using a 3-step enzymatic digestion protocol carried out in the absence of dithiothreitol, we demonstrate that DNA can be efficiently released from cells or tissues preserved in buffered formalin or the alcohol-based fixative GenoFix. This preparatory procedure can then be integrated to traditional phenol/chloroform extraction, a modified manual DNA IQ or automated DNA IQ/Te-Shake-based extraction in order to recover DNA for downstream applications. Quantitative recovery of high quality DNA was best achieved from specimens archived in GenoFix and extracted using magnetic bead capture.
存档组织在固定剂中保存，这是组织学检查、分子诊断程序以及法医调查中 DNA 分型分析的宝贵资源。然而，可用的材料通常在大小和数量上都受到限制。此外，由于甲醛（最常用的固定剂）产生的共价 DNA-蛋白质交联的存在，DNA 的回收常常受到严重损害。我们描述了缓冲液配方、样品裂解方案和 DNA 回收策略的评估，并定义了用于提取适合分子诊断和基因分型的高质量 DNA 的优化手动和自动化程序。使用在没有二硫苏糖醇的情况下进行的 3 步酶消化方案，我们证明可以从保存在缓冲甲醛或基于酒精的固定剂 GenoFix 中的细胞或组织中有效地释放 DNA。然后，可以将该预备程序集成到传统的苯酚/氯仿提取、改良的手动 DNA IQ 或基于自动 DNA IQ/Te-Shake 的提取中，以回收用于下游应用的 DNA。从使用磁性珠捕获提取的 GenoFix 中保存的标本中获得了最高质量的高定量回收 DNA。
