Department of Biological Sciences, Institute of Environmental Sustainability, Swansea University, Swansea SA2 8PP, UK, Bird Group, Department of Zoology, The Natural History Museum, Tring HP23 6AP, Herts, UK.
Mol Ecol Resour. 2008 May;8(3):551-60. doi: 10.1111/j.1471-8286.2007.02042.x.
We present a comprehensive protocol for extracting DNA from egg membranes and other internal debris recovered from the interior of blown museum bird eggs. A variety of commercially available DNA extraction methods were found to be applicable. DNA sequencing of polymerase chain reaction (PCR) products for a 176-bp fragment of mitochondrial DNA was successful for most egg samples (> 78%) even though the amount of DNA extracted (mean = 14.71 ± 4.55 ng/µL) was significantly less than that obtained for bird skin samples (mean = 67.88 ± 4.77 ng/µL). For PCR and sequencing of snipe (Gallinago) DNA, we provide eight new primers for the 'DNA barcode' region of COI mtDNA. In various combinations, the primers target a range of PCR products sized from 72 bp to the full 'barcode' of 751 bp. Not all possible combinations were tested with archive snipe DNA, but we found a significantly better success rate of PCR amplification for a shorter 176-bp target compared with a larger 288-bp fragment (67% vs. 39%). Finally, we explored the feasibility of whole genome amplification (WGA) for extending the use of archive DNA in PCR and sequencing applications. Of two WGA approaches, a PCR-based method was found to be able to amplify whole genomic DNA from archive skins and eggs from museum bird collections. After WGA, significantly more archive egg samples produced visible PCR products on agarose (56.9% before WGA vs. 79.0% after WGA). However, overall sequencing success did not improve significantly (78.8% compared with 83.0%).
我们提出了一个从博物馆吹制鸟蛋内部回收的卵膜和其他内部碎片中提取 DNA 的综合方案。发现多种市售 DNA 提取方法均可适用。尽管提取的 DNA 量（平均值= 14.71 ± 4.55ng/µL）明显少于鸟类皮肤样本（平均值= 67.88 ± 4.77ng/µL），但对大多数蛋样进行聚合酶链反应（PCR）产物的 176-bp 线粒体 DNA 测序仍然成功。对于 snipe（Gallinago）DNA 的 PCR 和测序，我们提供了 COI mtDNA 的“DNA 条码”区域的 8 个新引物。在各种组合中，引物的目标是一系列大小从 72bp 到全长“条码”751bp 的 PCR 产物。并非所有可能的组合都用档案 snipe DNA 进行了测试，但我们发现与较大的 288-bp 片段（67% 对 39%）相比，较短的 176-bp 目标的 PCR 扩增成功率显著提高。最后，我们探索了全基因组扩增（WGA）在扩展 PCR 和测序应用中使用档案 DNA 的可行性。在两种 WGA 方法中，发现基于 PCR 的方法能够从博物馆鸟类收藏的档案皮肤和卵中扩增全基因组 DNA。WGA 后，在琼脂糖上产生可见 PCR 产物的档案卵样本显著增加（WGA 前为 56.9%，WGA 后为 79.0%）。然而，总体测序成功率并没有显著提高（78.8%比 83.0%）。
