BRANCH CAPTURE REACTION IN PHYSICAL MAPPING

“自下而上”物理测绘的一个主要挑战是填补空白 重叠群之间。我们将应用分支捕获反应(BCR)，它是 在我们实验室开发，以准备丰富的库，以满足所需 克隆人。染色体行走文库将使用多个 方法，以便可以同时填补多个空白。 BCR使DNA部分双链能够与更长的， 对限制的修饰、置换链和更短的连接链 受体双链DNA的酶裂解位点。他们的反应是 序列特定且严格，附件要求 移位体和受体DNA双链的单链部分。 BCR可用于在电泳前标记DNA片段、标记A 用于亲和层析的片段，或同时引入可选择标记 以及与不同限制条件兼容的新的悬垂序列 克隆载体中的核酸内切酶位点。 具体来说，BCR和其他最先进的方法将应用于 获得高分辨率的人类9号染色体物理图谱，包括 有序克隆和一组有序的唯一序列标记的站点 (STS)。BCR方法将可用于其他 人类的染色体。

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.