Kemp Brian M, Winters Misa, Monroe Cara, Barta Jodi Lynn
1 Department of Anthropology, Washington State University, Pullman, Washington.
Hum Biol. 2014 Fall;86(4):313-29. doi: 10.13110/humanbiology.86.4.0313.
The success in recovering genetic profiles from aged and degraded biological samples is diminished by fundamental aspects of DNA extraction, as well as its long-term preservation, that are not well understood. While numerous studies have been conducted to determine whether one extraction method was superior to others, nearly all of them were initiated with no knowledge of the actual starting DNA quantity in the samples prior to extraction, so they ultimately compared the outcome of all methods relative to the best. Using quantitative PCR to estimate the copy count of synthetic standards before (i.e., "copies in") and after (i.e., "copies out") purification by the Qiagen MinElute PCR Purification Kit, we documented DNA loss within a pool of 16 different-sized fragments ranging from 106 to 409 bp in length, corresponding to those targeted by the PowerPlex 16 System (Promega, Madison, WI). Across all standards from 10(4) to 10(7) copies/μL, loss averaged between 21.75% and 60.56% (mean, 39.03%), which is not congruent with Qiagen's claim that 80% of 70 bp to 4 kb fragments are retained using this product (i.e., 20% loss). Our study also found no clear relationship either between DNA strand length and retention or between starting copy number and retention. This suggests that there is no molecule bias across the MinElute column membrane and highlights the need for manufacturers to clearly and accurately describe on what their claims are based, and should also encourage researchers to document DNA retention efficiencies of their own methods and protocols. Understanding how and where to reduce loss of molecules during extraction and purification will serve to generate clearer and more accurate data, which will enhance the utility of ancient and low-copy-number DNA as a tool for closing forensic cases or in reconstructing the evolutionary history of humans and other organisms.
从老化和降解的生物样本中恢复基因图谱的成功率，会因DNA提取及其长期保存等尚未被充分理解的基本因素而降低。虽然已经进行了大量研究来确定一种提取方法是否优于其他方法，但几乎所有这些研究在开始时都不知道提取前样本中实际的起始DNA量，所以它们最终是将所有方法的结果与最佳结果进行比较。我们使用定量PCR来估计通过Qiagen MinElute PCR纯化试剂盒纯化之前（即“纯化前拷贝数”）和之后（即“纯化后拷贝数”）合成标准品的拷贝数，记录了长度从106到409 bp的16种不同大小片段混合样本中的DNA损失，这些片段对应于PowerPlex 16系统（Promega公司，威斯康星州麦迪逊）所针对的片段。在所有浓度从10⁴到10⁷拷贝/μL的标准品中，损失平均在21.75%至60.56%之间（平均为39.03%），这与Qiagen公司声称使用该产品可保留70 bp至4 kb片段的80%（即20%损失）不一致。我们的研究还发现，DNA链长度与保留率之间以及起始拷贝数与保留率之间均无明显关系。这表明在MinElute柱膜上不存在分子偏好，并突出了制造商需要清晰准确地描述其声明的依据，同时也应鼓励研究人员记录自己方法和方案的DNA保留效率。了解在提取和纯化过程中如何以及在何处减少分子损失，将有助于生成更清晰、更准确的数据，这将提高古代和低拷贝数DNA作为解决法医案件或重建人类及其他生物进化历史工具的效用。
