Université Bordeaux 1, UMR 5199 PACEA, Laboratoire d'Anthropologie des Populations du Passé, Avenue des Facultés, 33405 Talence Cedex,
Forensic Sci Int. 2011 Jul 15;210(1-3):102-9. doi: 10.1016/j.forsciint.2011.02.010. Epub 2011 Mar 1.
The analysis of DNA from archaeological human remains is plagued by a unique set of methodological problems concerning contamination with modern exogenous DNA. Through an original approach, we propose complementary methods to identify all potential sources of contamination and complete guidelines for the validation of ancient human sequences. The study presented was conducted on non-European human samples (Polynesian and Amerindian) which were collected with all precautions during excavation. This permitted us to distinguish without ambiguity authentic and contaminant sequences. The samples' origins and histories were perfectly known, allowing us to trace all potential contamination sources and to determine the efficiency of precautions followed during all steps of the study. The data obtained confirm that precautions taken during sampling effectively prevent contamination. However, we demonstrate that human contamination can also be introduced during genetic analyses even if all precautions are strictly followed. Indeed, numerous human contaminations were detected in template-PCR products and negative controls, resulting in a striking diversity of contaminant mitochondrial DNA sequences. We argue that this contamination partly derives from the primers. To our knowledge, no previous experiment has been performed to investigate primers as a possible source of human contamination despite the fact that this specific type of contamination poses a real problem in terms of validating ancient human DNA studies. Finally, we confirm that the detection of contaminants in negative controls is clearly related to the number of PCR cycles used. This study enhances our understanding of contamination processes and confirms that, in reality, an absolutely contamination-free situation cannot be obtained. As a consequence, we propose improvements to the guidelines usually followed in the field in order to take the highly probable contamination of PCR reagents, including primers, into account.
对考古人类遗骸中的 DNA 进行分析时，会受到与现代外源性 DNA 污染相关的一系列独特方法问题的困扰。通过一种原创方法，我们提出了识别所有潜在污染源的补充方法，并为验证古代人类序列制定了完整的指南。本研究针对非欧洲人类样本（波利尼西亚和美洲印第安人）进行，这些样本在挖掘过程中采取了所有预防措施进行收集。这使我们能够毫不含糊地区分真实序列和污染序列。样本的来源和历史都非常清楚，这使我们能够追踪所有潜在的污染来源，并确定在研究的所有步骤中所采取的预防措施的效率。获得的数据证实，采样过程中采取的预防措施可以有效防止污染。然而，我们证明，即使严格遵循所有预防措施，在遗传分析过程中也可能引入人类污染。事实上，在模板-PCR 产物和阴性对照中检测到了许多人类污染，导致污染的线粒体 DNA 序列呈现出惊人的多样性。我们认为，这种污染部分源自引物。据我们所知，尽管这种特定类型的污染在验证古代人类 DNA 研究方面确实存在问题，但之前没有进行过任何实验来研究引物是否可能成为人类污染的来源。最后，我们证实，阴性对照中污染物的检测与使用的 PCR 循环数明显相关。本研究增强了我们对污染过程的理解，并证实，实际上，无法达到绝对无污染的情况。因此，我们建议改进该领域通常遵循的指南，以考虑到高度可能污染的 PCR 试剂，包括引物。
