Loreille Odile M, Diegoli Toni M, Irwin Jodi A, Coble Michael D, Parsons Thomas J
Armed Forces DNA Identification Laboratory, 1413 Research Blvd., Bldg. 101, Rockville, MD 20850, United States.
Forensic Sci Int Genet. 2007 Jun;1(2):191-5. doi: 10.1016/j.fsigen.2007.02.006. Epub 2007 Mar 12.
In historical cases, missing persons' identification, mass disasters, and ancient DNA investigations, bone and teeth samples are often the only, and almost always the best, biological material available for DNA typing. This is because of the physical and chemical barrier that the protein:mineral matrix of bone poses to environmental deterioration and biological attack. Most bone extraction protocols utilized in the forensic community involve an incubation period of bone powder in extraction buffer for proteinase digestion, followed by the collection of the supernatant, and the disposal of large quantities of undissolved bone powder. Here we present an extremely efficient protocol for recovery of DNA by complete demineralization, resulting in full physical dissolution of the bone sample. This is performed in a manner that retains and concentrates all the reagent volume, for complete DNA recovery. For our study, we selected 14 challenging bone samples. The bones were extracted side-by-side with our new demineralization protocol and the standard extraction protocol in use at AFDIL. A real-time quantification assay based on the amplification of a 143 bp mtDNA fragment showed that this new demineralization protocol significantly enhances the quantity of DNA that can be extracted and amplified from degraded skeletal remains. We have used this technique to successfully recover authentic DNA sequences from extremely challenging samples that failed repeatedly using the standard protocol.
在历史案件、失踪人员身份鉴定、大规模灾难及古DNA研究中，骨骼和牙齿样本往往是唯一且几乎总是最佳的可用于DNA分型的生物材料。这是因为骨骼的蛋白质：矿物质基质对环境恶化和生物攻击构成了物理和化学屏障。法医界使用的大多数骨骼提取方案都包括将骨粉在提取缓冲液中孵育以进行蛋白酶消化，随后收集上清液，并处理大量未溶解的骨粉。在此，我们提出了一种通过完全脱矿质来回收DNA的极其有效的方案，从而使骨样本完全物理溶解。该过程以保留和浓缩所有试剂体积的方式进行，以实现DNA的完全回收。在我们的研究中，我们选择了14个具有挑战性的骨骼样本。这些骨骼与我们新的脱矿质方案以及AFDIL正在使用的标准提取方案并行进行提取。基于143 bp线粒体DNA片段扩增的实时定量分析表明，这种新的脱矿质方案显著提高了从降解的骨骼遗骸中可提取和扩增的DNA数量。我们已使用该技术从使用标准方案反复失败的极具挑战性的样本中成功回收了真实的DNA序列。
