一种基于抗生物素蛋白-生物素相互作用的基因富集方法。应用于果蝇核糖体RNA基因。

A method for gene enrichment based on the avidin-biotin interaction. Application to the Drosophila ribosomal RNA genes.

作者信息

Manning J, Pellegrini M, Davidson N

出版信息

Biochemistry. 1977 Apr 5;16(7):1364-70. doi: 10.1021/bi00626a020.

PMID:191068

Abstract

A method of enriching, from the total DNA of an organism, for long DNA strands carrying a particular gene is described. The purified RNA corresponding to the gene is covalently attached to biotin via a cytochrome c bridge. This modified RNA is hybridized to the total DNA. Those DNA strands which hybridize are separated from all the other DNA, using the avidin-biotin interaction, by one of two methods. Avidin is covalently attached to submicroscopic polymer spheres; the complexes of avidin spheres with the DNA: RNA-biotin hybrids band in CsCl at a much lower buoyant density than does free DNA. Alternatively, the DNA:RNA-biotin hybrids are isolated by affinity chromatography on an avidin-solid support column. These methods have been used to prepare long single strands of Drosophila ribosomal DNA (rDNA) in high yield and 42 to 80% pure.

摘要

描述了一种从生物体的总DNA中富集携带特定基因的长DNA链的方法。与该基因对应的纯化RNA通过细胞色素c桥共价连接到生物素上。这种修饰的RNA与总DNA杂交。利用抗生物素蛋白-生物素相互作用，通过两种方法之一将杂交的DNA链与所有其他DNA分离。抗生物素蛋白共价连接到亚微观聚合物球体上；抗生物素蛋白球体与DNA:RNA-生物素杂交体的复合物在CsCl中的浮力密度比游离DNA低得多。或者，通过在抗生物素蛋白固相支持柱上进行亲和层析来分离DNA:RNA-生物素杂交体。这些方法已被用于高产制备果蝇核糖体DNA（rDNA）的长单链，纯度为42%至80%。