International Commission on Missing Persons, Alipasina 45a, Sarajevo, Bosnia and Herzegovina.
Forensic Sci Int Genet. 2012 May;6(3):398-406. doi: 10.1016/j.fsigen.2011.08.004. Epub 2011 Aug 31.
During the 7 year period from 2002 to 2009 a high volume, silica-binding DNA extraction protocol for bone, based on modified QIAGEN's Blood Maxi Kit protocol was highly successful permitting the DNA matching of >14,500 missing persons from former Yugoslavia. This method, however, requires large amount of bone material and large volumes of reagents. The logical evolution was to develop a more efficient extraction protocol for bone samples that uses significantly less starting material while increasing the success in obtaining DNA results from smaller, more challenging samples. In this study we compared the performance of ICMP's original protocol against an automatable full demineralization approach. In order to provide reliable results and to simulate a wide variety of cases, we analyzed 40 bone samples in a comparative study based on DNA concentrations and quality of resulting STR profiles. The new protocol results in the dissolution of the entire bone powder sample, thus eliminating the possibility that DNA is left behind, locked in remaining solid bone matrix. For the majority of samples tested, the DNA concentrations obtained from half a gram of fully digested bone material were equivalent to or greater than the ones obtained from 2g of partially demineralized bone powder. Furthermore, the full demineralization process significantly increases the proportion of full profiles reflecting the correlation with better DNA quality. This method has been adapted for the QIAcube robotic platform. The performance of this automated full demineralization protocol is similar to the manual version and increases overall lab throughput. It also simplifies the process by eliminating quality control procedures that are advisable in manual procedures, and overall reduces the chance of human error. Finally we described a simple and efficient post-extraction clean-up method that can be applied to DNA extracts obtained from different protocols. This protocol has also been adjusted for the QIAcube platform.
在 2002 年至 2009 年的 7 年期间，我们成功地建立了一种基于 QIAGEN Blood Maxi 试剂盒改良版的高容量、结合硅的骨骼 DNA 提取方案，该方案允许对来自前南斯拉夫的 14500 多名失踪人员进行 DNA 匹配。然而，这种方法需要大量的骨骼材料和大量的试剂。逻辑上的发展是开发一种更有效的骨骼样本提取方案，该方案使用的起始材料更少，同时增加从小而更具挑战性的样本中获得 DNA 结果的成功率。在这项研究中，我们比较了 ICMP 原始方案和自动化完全脱矿化方法的性能。为了提供可靠的结果并模拟各种情况，我们在基于 DNA 浓度和所得 STR 图谱质量的比较研究中分析了 40 个骨骼样本。新方案导致整个骨粉样本溶解，从而消除了 DNA 残留在剩余的固体骨基质中的可能性。对于大多数测试的样本，从半克完全消化的骨骼材料中获得的 DNA 浓度与从 2 克部分脱矿化的骨粉中获得的浓度相当或更高。此外，完全脱矿化过程显著增加了完整图谱的比例，反映了与更好的 DNA 质量的相关性。该方法已被改编为 QIAcube 机器人平台。这种自动化完全脱矿化方案的性能与手动版本相似，并提高了整体实验室吞吐量。它还通过消除在手动程序中建议的质量控制程序来简化该过程，并总体上减少人为错误的机会。最后，我们描述了一种简单有效的提取后清洁方法，可应用于从不同方案获得的 DNA 提取物。该方案也已针对 QIAcube 平台进行了调整。
