Griffin Jeanine
Andrology and IVF Laboratories, Division of Urology, Department of Surgery, University of Utah School of Medicine, Salt Lake City, UT, USA.
Methods Mol Biol. 2013;927:379-84. doi: 10.1007/978-1-62703-038-0_32.
High quality DNA extractions developed for mammalian somatic cells are ineffective for sperm, due mainly to the high degree of nuclear compaction in sperm. The highly specialized nuclear proteins in sperm create a chromatin structure that is at least six times denser than histone bound DNA. Unlike somatic cells, sperm DNA is highly compacted by the replacement of histones with sperm-specific low molecular weight proteins called protamines. Both the protamines and the disulfide bridges formed within and between protamines inhibit the extraction of sperm DNA by standard techniques used for somatic cells. Here we describe the guanidine thiocyanate method reported by Hossain with additional modifications resulting in high molecular weight DNA of high quality with an A260/280 ratio ranging between 1.8 and 2.0 and an A260/230 ratio of 2.0 and greater. The DNA is efficiently digested with restriction enzymes and amplified by PCR.
为哺乳动物体细胞开发的高质量DNA提取方法对精子无效，主要原因是精子中的核高度致密。精子中高度特化的核蛋白形成了一种染色质结构，其密度比组蛋白结合的DNA至少高六倍。与体细胞不同，精子DNA通过被称为鱼精蛋白的精子特异性低分子量蛋白取代组蛋白而高度致密。鱼精蛋白以及鱼精蛋白内部和之间形成的二硫键都抑制了用体细胞标准技术提取精子DNA。在此，我们描述了Hossain报道的硫氰酸胍方法，并进行了额外改进，得到了高质量的高分子量DNA，其A260/280比值在1.8至2.0之间，A260/230比值为2.0或更高。该DNA能用限制性内切酶有效消化并通过PCR扩增。
