Max Planck Institute for Evolutionary Anthropology, Deutscher Platz 6, D-04103 Leipzig, Germany Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA Department of Biology, University of York, YO10 5YW, York, UK.
Mol Ecol Resour. 2010 Jul;10(4):677-83. doi: 10.1111/j.1755-0998.2009.02824.x. Epub 2009 Dec 29.
Genetic analyses using museum specimens and ancient DNA from fossil samples are becoming increasingly important in phylogenetic and especially population genetic studies. Recent progress in ancient DNA sequencing technologies has substantially increased DNA sequence yields and, in combination with barcoding methods, has enabled large-scale studies using any type of DNA. Moreover, more and more studies now use nuclear DNA sequences in addition to mitochondrial ones. Unfortunately, nuclear DNA is, due to its much lower copy number in living cells compared to mitochondrial DNA, much more difficult to obtain from low-quality samples. Therefore, a DNA extraction method that optimizes DNA yields from low-quality samples and at the same time allows processing many samples within a short time frame is immediately required. In fact, the major bottleneck in the analysis process using samples containing low amounts of degraded DNA now lies in the extraction of samples, as column-based methods using commercial kits are fast but have proven to give very low yields, while more efficient methods are generally very time-consuming. Here, we present a method that combines the high DNA yield of batch-based silica extraction with the time-efficiency of column-based methods. Our results on Pleistocene cave bear samples show that DNA yields are quantitatively comparable, and in fact even slightly better than with silica batch extraction, while at the same time the number of samples that can conveniently be processed in parallel increases and both bench time and costs decrease using this method. Thus, this method is suited for harvesting the power of high-throughput sequencing using the DNA preserved in the millions of paleontological and museums specimens.
利用博物馆标本和化石样本中的古代 DNA 进行遗传分析，在系统发育学，特别是种群遗传学研究中变得越来越重要。最近，古代 DNA 测序技术的进步大大提高了 DNA 序列的产量，并且与条形码方法相结合，使得使用任何类型的 DNA 进行大规模研究成为可能。此外，现在越来越多的研究除了线粒体 DNA 之外还使用核 DNA 序列。不幸的是，由于核 DNA 在活细胞中的拷贝数比线粒体 DNA 低得多，因此从低质量样本中获取核 DNA 要困难得多。因此，立即需要一种能够从低质量样本中优化 DNA 产量，同时允许在短时间内处理大量样本的 DNA 提取方法。事实上，使用含有低量降解 DNA 的样本进行分析的主要瓶颈现在在于样本的提取，因为使用商业试剂盒的基于柱的方法速度很快，但已被证明产量非常低，而更有效的方法通常非常耗时。在这里，我们提出了一种将批量硅基提取的高 DNA 产量与基于柱的方法的高效性相结合的方法。我们对更新世洞穴熊样本的研究结果表明，DNA 产量在定量上是可比的，实际上甚至比硅基批量提取略好，同时，使用这种方法可以方便地同时处理更多的样本，并且减少了实验台时间和成本。因此，这种方法非常适合利用保存在数百万个古生物学和博物馆标本中的 DNA 来实现高通量测序的优势。
