van Brabant A J, Hunt S Y, Fangman W L, Brewer B J
Department of Genetics, University of Washington, Seattle 98195-7360, USA.
Electrophoresis. 1998 Jun;19(8-9):1239-46. doi: 10.1002/elps.1150190803.
DNA fragments that contain an active origin of replication generate bubble-shaped replication intermediates with diverging forks. We describe two methods that use two-dimensional (2-D) agarose gel electrophoresis along with DNA sequence information to identify replication origins in natural and artificial Saccharomyces cerevisiae chromosomes. The first method uses 2-D gels of overlapping DNA fragments to locate an active chromosomal replication origin within a region known to confer autonomous replication on a plasmid. A variant form of 2-D gels can be used to determine the direction of fork movement, and the second method uses this technique to find restriction fragments that are replicated by diverging forks, indicating that a bidirectional replication origin is located between the two fragments. Either of these two methods can be applied to the analysis of any genomic region for which there is DNA sequence information or an adequate restriction map.
含有活性复制起点的DNA片段会产生带有叉状延伸的泡状复制中间体。我们描述了两种方法，这两种方法利用二维（2-D）琼脂糖凝胶电泳以及DNA序列信息来鉴定天然和人工酿酒酵母染色体中的复制起点。第一种方法使用重叠DNA片段的二维凝胶，以在已知能赋予质粒自主复制能力的区域内定位活性染色体复制起点。二维凝胶的一种变体形式可用于确定叉状移动的方向，第二种方法使用该技术来寻找由叉状延伸复制的限制性片段，这表明在两个片段之间存在双向复制起点。这两种方法中的任何一种都可应用于对有DNA序列信息或足够限制酶切图谱的任何基因组区域进行分析。
