Mohandesan Elmira, Prost Stefan, Hofreiter Michael
Allan Wilson Centre for Molecular Ecology and Evolution, Institute of Natural Sciences, Massey University, 102904, NSMC, Auckland, New Zealand.
Methods Mol Biol. 2012;840:101-10. doi: 10.1007/978-1-61779-516-9_14.
A major challenge for ancient DNA (aDNA) studies using museum specimens is that sampling procedures usually involve at least the partial destruction of each specimen used, such as the removal of skin, pieces of bone, or a tooth. Recently, a nondestructive DNA extraction method was developed for the extraction of amplifiable DNA fragments from museum specimens without appreciable damage to the specimen. Here, we examine the utility of this method by attempting DNA extractions from historic (older than 70 years) chimpanzee specimens. Using this method, we PCR-amplified part of the mitochondrial HVR-I region from 65% (56/86) of the specimens from which we attempted DNA extraction. However, we found a high incidence of multiple sequences in individual samples, suggesting substantial cross-contamination among samples, most likely originating from storage and handling in the museums. Consequently, reproducible sequences could be reconstructed from only 79% (44/56) of the successfully extracted samples, even after multiple extractions and amplifications. This resulted in an overall success rate of just over half (44/86 of samples, or 51% success), from which 39 distinct HVR-I haplotypes were recovered. We found a high incidence of C to T changes, arguing for both low concentrations of and substantial damage to the endogenous DNA. This chapter highlights both the potential and the limitations of nondestructive DNA extraction from museum specimens.
利用博物馆标本进行古DNA（aDNA）研究面临的一个主要挑战是，采样程序通常至少会对所使用的每个标本造成部分破坏，比如去除皮肤、骨块或一颗牙齿。最近，一种非破坏性DNA提取方法被开发出来，用于从博物馆标本中提取可扩增的DNA片段，且不会对标本造成明显损害。在此，我们通过尝试从历史悠久（超过70年）的黑猩猩标本中提取DNA来检验该方法的实用性。使用这种方法，我们从65%（56/86）尝试进行DNA提取的标本中PCR扩增出了线粒体高变区I（HVR-I）区域的部分片段。然而，我们发现单个样本中存在多个序列的情况很常见，这表明样本之间存在大量交叉污染，很可能源于博物馆中的储存和处理过程。因此，即使经过多次提取和扩增，也只能从79%（44/56）成功提取的样本中重建出可重复的序列。这导致总体成功率略高于一半（86个样本中的44个，即51%的成功率），从中 recovered 39种不同的HVR-I单倍型。我们发现C到T的变化发生率很高，这表明内源性DNA浓度低且受到了严重损伤。本章强调了从博物馆标本中进行非破坏性DNA提取的潜力和局限性。 （注：原文中“from which 39 distinct HVR-I haplotypes were recovered”这里的“recovered”翻译为“恢复”不太准确，可根据语境灵活调整为如“获得”等，但按照要求未做修改。）
