State of California, Department of Justice, Jan Bashinski DNA Laboratory, 1001 W. Cutting Blvd., Richmond, CA 94804, USA.
State of California, Department of Justice, Jan Bashinski DNA Laboratory, 1001 W. Cutting Blvd., Richmond, CA 94804, USA.
Forensic Sci Int Genet. 2018 May;34:170-177. doi: 10.1016/j.fsigen.2018.02.014. Epub 2018 Feb 21.
The efficacy of a DNA differential extraction procedure relies on reducing the amount of non-sperm female DNA carryover into the sperm fraction, while providing a sufficient recovery of male DNA from the sperm cell component. A standard approach to this extraction is to use a mild initial lysis step to digest the female (epithelial cell) component in the mixture, followed by a series of centrifugation and wash steps to further purify the resulting sperm-pellet fraction. This sperm fraction is then digested in the presence of a chemical reducing agent in preparation for DNA extraction. This method has been employed with relatively few changes since its introduction in the mid-1980s, despite numerous attempts to develop new or improved procedures. In this report, we demonstrate that it is possible to improve the efficacy of the standard differential extraction by applying simple modifications that can reduce the amount of female DNA carryover into the sperm fraction, with no adverse effects on the recovery of male DNA. In one modification, the addition of a second mild lysis step at the beginning of the differential extraction procedure improved the average male-to-female DNA ratio in the sperm fraction by 3- to 6-fold. In another modification, a "tube transfer" step was added to move the re-suspended sperm pellet to a new tube for the second mild lysis and subsequent wash steps. With this modification, the average male-to-female DNA ratio in the sperm fraction was improved by 4- to 90-fold, relative to results obtained for the non-modified differential extraction method. These modifications may be accomplished using tools and reagents that are already present in most forensic DNA laboratories, so that implementation should be relatively low-cost and practical.
一种 DNA 差异提取程序的功效依赖于减少非精子女性 DNA 转移到精子部分的量，同时从精子细胞成分中提供足够的男性 DNA 回收。这种提取的标准方法是使用温和的初始裂解步骤来消化混合物中的女性（上皮细胞）成分，然后进行一系列离心和洗涤步骤，以进一步纯化所得的精子沉淀部分。然后，在化学还原剂的存在下，将该精子部分消化，为 DNA 提取做准备。自 20 世纪 80 年代中期推出以来，尽管多次尝试开发新的或改进的程序，但这种方法仅进行了相对较少的改变。在本报告中，我们证明通过应用简单的修改可以提高标准差异提取的功效，这些修改可以减少女性 DNA 转移到精子部分的量，而对男性 DNA 的回收没有不利影响。在一种改进中，在差异提取程序的开始时添加第二个温和的裂解步骤，可将精子部分中男性到女性 DNA 的平均比值提高 3 至 6 倍。在另一种改进中，添加了“管转移”步骤，将重新悬浮的精子沉淀转移到新管中，进行第二次温和的裂解和随后的洗涤步骤。通过这种改进，精子部分中男性到女性 DNA 的平均比值提高了 4 至 90 倍，与非修饰的差异提取方法相比。这些修改可以使用大多数法医 DNA 实验室已经存在的工具和试剂来完成，因此实施应该相对低成本和实用。
