A method for preparing genomic DNA that restrains branch migration of Holliday junctions.
A method for preparing genomic DNA that restrains branch migration of Holliday junctions.
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DOI：
10.1093/nar/28.2.e6
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发表时间：
2000-01-15
影响因子：
14.9
通讯作者：
Lichten, M
中科院分区：
文献类型：
--
作者：
Allers, T;Lichten, M
Holliday连接是基因重组的中心中间体。这种四链DNA结构能够自发地进行分支迁移，并在标准的DNA提取方案中丢失。为了分离和鉴定含有Holliday连接的重组中间体，我们开发了一种抑制四向DNA连接分支迁移的快速方案。阳离子洗涤剂十六烷基三甲基溴化铵用于裂解细胞和沉淀DNA。操纵是在阳离子六价钴(III)或镁的存在下进行的，这两种阳离子在分支迁移非常缓慢的堆积-X构型中稳定Holliday结。该协议使用一种敏感的自发分支迁移试验进行了评估，并被证明保存了人工Holliday连接和包含四向连接的减数分裂重组中间产物。
The Holliday junction is a central intermediate in genetic recombination. This four-stranded DNA structure is capable of spontaneous branch migration, and is lost during standard DNA extraction protocols. In order to isolate and characterize recombination intermediates that contain Holliday junctions, we have developed a rapid protocol that restrains branch migration of four-way DNA junctions. The cationic detergent hex-adecyltrimethylammonium bromide is used to lyse cells and precipitate DNA. Manipulations are performed in the presence of the cations hexamine cobalt(III) or magnesium, which stabilize Holliday junctions in a stacked-X configuration that branch migrates very slowly. This protocol was evaluated using a sensitive assay for spontaneous branch migration, and was shown to preserve both artificial Holliday junctions and meiotic recombination intermediates containing four-way junctions.