BRANCH CAPTURE REACTION IN PHYSICAL MAPPING

“自下而上”物理制图的一个主要挑战是填补空白 在重叠群之间。我们将应用分支捕获反应（BCR）， 在我们的实验室开发，准备丰富的图书馆，为所需的 克隆 染色体步移文库将使用多重 可以同时填充多个间隙。 BCR能够使DNA部分双链体与较长的， 修饰的置换链和较短的连接链 受体双链体DNA的酶切割位点。 反应 序列特异性和严格性，连接需要同源性， 置换体的单链部分和受体DNA双链体。 BCR可用于在电泳前标记DNA片段，标记DNA片段， 片段进行亲和层析，或引入选择性标记 和与不同限制相容的新突出端序列 克隆载体中的核酸内切酶位点。 具体而言，BCR和其他最先进的方法将应用于 获得人类9号染色体的高分辨率物理图谱，包括 有序克隆和一组有序的独特序列标记位点 （STS）。 BCR方法将可用于其他合作研究 人类染色体

A major challenge in "bottom-up" physical mapping is filling in gaps between contigs. We will apply the Branch Capture Reaction (BCR), which was developed in our laboratory, to prepare libraries enriched for the desired clones. Chromosome-walking libraries will be constructed using multiplex methods so that many gaps may be filled at the same time. BCR enables covalent attachment of a DNA partial duplex with a longer, modified, displacer strand and a shorter linker strand to a restriction enzyme cleavage site of a recipient duplex DNA. The reaction is sequence-specific and stringent, with attachment requiring homology between the single-stranded portion of the displacer and the recipient DNA duplex. BCR may be used to label a DNA fragment prior to electrophoresis, mark a fragment for affinity chromatography, or introduce both a selectable marker and a new overhang sequence compatible with a different restriction endonuclease site in a cloning vector. Specifically, BCR and other state-of-the-art methods will be applied toward obtaining a high resolution physical map of human chromosome 9, including both ordered clones and an ordered set of unique sequence tagged sites (STS). The BCR method will be available for collaborative studies of other human chromosomes.