Ripoll P J, O'Sullivan D M, Edwards K J, Rodgers M
University of Bristol, Long Ashton, UK.
Biotechniques. 2000 Aug;29(2):271-4, 276. doi: 10.2144/00292st02.
Bacterial artificial chromosome (BAC) libraries are an important tool for positional cloning, gene analysis and physical mapping. During studies using BAC clones, it is often necessary to organize them into contiguous sequences (contigs). To finalize, join and extend the contigs, both cloning and sequencing of the ends of the inserts are required. Here, we describe a low-cost, accessible, fast and powerful method for the routine isolation of BAC ends. This method allows the isolation of 20 BAC clone ends in one day. The analysis of the ends reveals fragment sizes compatible with sequencing, and the structure of these clones allows the sequencing of both ends using the same plasmid. Moreover, long end fragments can be sequenced in both directions.
细菌人工染色体（BAC）文库是用于定位克隆、基因分析和物理图谱构建的重要工具。在使用BAC克隆的研究过程中，常常需要将它们组装成连续序列（重叠群）。为了完成、连接和扩展重叠群，需要对插入片段的末端进行克隆和测序。在此，我们描述了一种低成本、易操作、快速且强大的方法，用于常规分离BAC末端。该方法可在一天内分离出20个BAC克隆末端。对末端的分析揭示了与测序兼容的片段大小，并且这些克隆的结构允许使用同一质粒对两端进行测序。此外，长末端片段可以双向测序。
