Ivessa Andreas S
Department of Cell Biology and Molecular Medicine, University of Medicine and Dentistry of New Jersey, Newark, NJ, USA.
Methods Mol Biol. 2013;1054:83-103. doi: 10.1007/978-1-62703-565-1_5.
The analysis of replication intermediates by the neutral-neutral two-dimensional agarose gel technique allows determining the chromosomal positions where DNA replication initiates, whether replication forks pause or stall at specific sites, or whether two DNA molecules undergo DNA recombination events. This technique does not, however, immediately tell in which direction replication forks migrate through the DNA region under investigation. Here, we describe the procedure to determine the direction of replication fork progression by carrying out a restriction enzyme digest of DNA imbedded in agarose after the completion of the first dimension of a 2D gel.
通过中性-中性二维琼脂糖凝胶技术分析复制中间体，能够确定DNA复制起始的染色体位置、复制叉是否在特定位点暂停或停滞，或者两个DNA分子是否发生DNA重组事件。然而，该技术并不能直接告知复制叉在被研究的DNA区域中沿哪个方向迁移。在此，我们描述了一种方法，即在二维凝胶的第一维电泳完成后，对包埋在琼脂糖中的DNA进行限制性酶切，以确定复制叉前进的方向。
