Rubenstein J L, Brice A E, Ciaranello R D, Denney D, Porteus M H, Usdin T B
Department of Psychiatry and Behavioral Sciences, Stanford Medical School, CA.
Nucleic Acids Res. 1990 Aug 25;18(16):4833-42. doi: 10.1093/nar/18.16.4833.
We describe a subtractive hybridization protocol which is designed to permit subtractions between cDNA libraries. The method uses single-stranded phagemids with directional inserts as both the driver and the target. We modified the M13 phagemid vector pBluescript for the directional cDNA cloning and subtractive hybridization. Two simplified methods for efficient construction of directional cDNA libraries are also described. Using a model system, we found that one round of subtractive hybridization results in a 5,000-fold specific subtraction of abundant molecules. We used two methods to quantify the efficiency and verify the specificity of the subtraction. In order to obtain these subtraction efficiencies, it was necessary to develop a method to purify the single-stranded DNA to homogeneity. The single-stranded purification involved using potassium iodide (KI) density centrifugation, restriction endonuclease digestion and phenol extraction in the presence of magnesium. We describe the several advantages of using directional inserts for the subtraction procedure.
我们描述了一种消减杂交方案，该方案旨在允许在cDNA文库之间进行消减。该方法使用带有定向插入片段的单链噬菌粒作为驱动子和靶标。我们对M13噬菌粒载体pBluescript进行了改造，用于定向cDNA克隆和消减杂交。还描述了两种高效构建定向cDNA文库的简化方法。使用一个模型系统，我们发现一轮消减杂交可导致丰富分子的5000倍特异性消减。我们使用两种方法来量化消减效率并验证消减的特异性。为了获得这些消减效率，有必要开发一种将单链DNA纯化至同质的方法。单链纯化包括使用碘化钾（KI）密度离心、限制性内切酶消化和在镁存在下的苯酚提取。我们描述了在消减过程中使用定向插入片段的几个优点。
