Modified guanidinium thiocyanate method for human sperm DNA isolation
Modified guanidinium thiocyanate method for human sperm DNA isolation
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DOI：
10.1093/molehr/3.11.953
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发表时间：
1997-11-01
影响因子：
4
通讯作者：
Thorneycroft, IH
中科院分区：
文献类型：
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作者：
Hossain, AM;Rizk, B;Thorneycroft, IH
哺乳动物精子染色质高度浓缩，因此从此类染色质中分离 DNA 可能是一项艰巨的任务。从体细胞产生高质量 DNA 的程序无法产生高质量的精子 DNA。在这项研究中，我们对以前使用的胍法进行了修改，使其能够简单有效地分离人类精子 DNA。在我们的方法中，裂解缓冲液含有胍、柠檬酸钠、肌氨酰、蛋白酶 K 和巯基乙醇。最初的胍方法中未使用蛋白酶 K，但已包含在我们的方案中。省略了原始程序中描述的裂解物的 CsCl 离心。相反，将异丙醇直接添加到裂解缓冲液中以收获 DNA。这种改良的胍方法产生了高分子量 DNA，而其他两种方法则导致大量 DNA 降解。 Southern 印迹分析表明，限制酶消化采用改良方法制备的 DNA 没有困难。由于改良胍方法是一种简单的一步程序，避免了均质、有机溶剂、离心，更重要的是，产生了无降解的 DNA，因此当需要来自成熟生殖细胞的 DNA 时，它可能是首选方法。
Mammalian sperm chromatin is highly condensed, so isolating DNA from such chromatin can be a formidable task. The procedures that produce high quality DNA from somatic cells fail to yield quality sperm DNA. In this study we have modified the previously used guanidinium method to make it simple and efficient in isolating human sperm DNA. In our method, the lysis buffer contained guanidinium, sodium citrate, sarkosyl, proteinase K and mercaptoethanol. Proteinase K was not used in the original guanidinium method but was included in our protocol. CsCl centrifugation of the lysate, as described in the original procedure, was omitted. Instead, isopropyl alcohol was added directly to the lysis buffer to harvest the DNA. This modified guanidinium method generated high molecular weight DNA while the other two methods resulted in considerable DNA degradation. There was no difficulty in restriction enzyme digestion of DNA prepared by the modified method as revealed by Southern blot analysis. Since the modified guanidinium method is a simple one-step procedure which avoids homogenization, organic solvents, centrifugation and, more importantly, produces degradation-free DNA, it could be the method of choice when DNA from mature germ cells is needed.