Lahiri D K, Bye S, Nurnberger J I, Hodes M E, Crisp M
Department of Psychiatry, Indiana University School of Medicine, Indianapolis 46202-4887.
J Biochem Biophys Methods. 1992 Dec;25(4):193-205. doi: 10.1016/0165-022x(92)90014-2.
We compared ten methods for extraction of DNA from whole blood. Nine methods require incubation with either enzymes or treatment of organic solvents or both. The 'Rapid Method' (RM) (Method 10) avoids the use of organic solvents (phenol/chloroform) and eliminates completely the use of proteinase K. Thus, the time and cost of DNA extraction are reduced significantly. This is accomplished by salting out and precipitation of the cellular proteins in saturated sodium chloride. This method takes less than an hour to completion, without compromising the yield or the quality of DNA. Using RM, we can make DNA from 0.1 ml of whole blood and as little as 0.5 ml of blood yields DNA sufficient to run a few Southern blots. The RM can also be applied to packed cells. The DNA is free of RNA, protein and degrading enzymes. The uncut DNA runs as a typical slow-migrating, high-molecular-weight and undegraded species in an agarose gel. The DNA is suitable for digestion by various restriction endonucleases. This procedure works equally well with fresh blood samples and with those that are stored at 4 degrees C and -70 degrees C. To our knowledge the RM reported here is the safest, fastest and most quantitative and economical method for preparation of DNA from whole blood and cells.
我们比较了从全血中提取DNA的十种方法。九种方法需要用酶孵育，或用有机溶剂处理，或两者兼用。“快速方法”（RM）（方法10）避免使用有机溶剂（苯酚/氯仿），并完全不用蛋白酶K。因此，DNA提取的时间和成本显著降低。这是通过在饱和氯化钠中盐析和沉淀细胞蛋白来实现的。该方法不到一小时就能完成，且不影响DNA的产量或质量。使用RM，我们可以从0.1毫升全血中提取DNA，仅0.5毫升血液产生的DNA就足以进行几次Southern印迹分析。RM也可应用于浓缩细胞。提取的DNA不含RNA、蛋白质和降解酶。未切割的DNA在琼脂糖凝胶中以典型的慢迁移、高分子量且未降解的条带形式出现。该DNA适合用各种限制性内切酶进行消化。此方法对新鲜血液样本以及保存在4℃和-70℃的样本同样有效。据我们所知，这里报道的RM是从全血和细胞中制备DNA最安全、最快、最定量且最经济的方法。
