EXTRACTING GENES FROM CLONED SEQUENCE DNA

为人类生成物理地图和重叠克隆集 基因组已经发展到YAC和粘粒克隆的重叠群 将在未来几年内用于大多数基因组区域。这个 基因图谱也得到了改进，因此现在相对简单 通过家族性研究定位疾病基因。这两个基因组图谱 在识别身体午睡基因的层面上，努力是一致的。 基因识别可以帮助确定大规模DNA的优先顺序 排序，可以提供起点和路标，以帮助 测序策略，并允许直接与病毒的基因组进行比较 模型生物。对于疾病基因识别，驱动力 许多人类基因研究，隔离的位置牵连 候选人是发现罪魁祸首缺陷的关键。饱和度 具有一个或两个基因序列的特定染色体区域 使用全球基因组不能有效地实现上述目的 映射策略。相反，它需要基因的密集应用。 识别--针对特定感兴趣区域的技术。 目前，有许多不同的战略，这些战略 提议完成这项任务，但这些都没有得到系统的 比较一下。 人类基因组中分析最深入的区域之一是条带 4p16.3，最近克隆的亨廷顿病基因。详细 遗传和物理图谱已经可用，YAC和粘粒重叠群已经 目前正在构建一个2Mb片段的完整DNA序列 已生成。我们建议利用这一地区来执行一项急需的 用基因饱和基因组区域的策略评估 序列和同源区域的详细比较 老鼠。我们将比较外显子扩增，基于 亲和力标记和基于序列的外显子预测 分离表达序列，同时使这段基因饱和 4p16.3，含基因。小鼠基因组的等同区域也将是 基因的含量、定位和基因组覆盖率将被克隆 与人类相比。这项研究将为以下工作提供宝贵的经验 将饱和策略应用于任何指定的基因组区域 分离疾病基因，以便于更详细的作图 战略，或在内部或之间建立结构性联系 物种。

The generation of physical maps and overlapping clone sets for the human genome has progressed to the point where contigs of YAC and cosmid clones will be available for most genomic regions within the next few years. The genetic map has also improved so that it is now relatively straightforward to localize disease genes by family studies. These two genome mapping efforts converge-at the level of identifying genes on the physical 'nap. Gene identification can help set priorities for large-scale DNA sequencing, can provide starting points and guideposts to aid in sequencing strategies and allows direct comparison with the genomes of model organisms. For disease gene identification, the driving force of much human genetic research, isolation of positionally implicated candidates is essential to discovering the culprit defect. The saturation of a particular chromosomal region with gene sequences for either or both of the above purposes is not efficiently achieved using a global genome mapping strategy. Rather, it requires the intensive application of gene identification -techniques to the particular region of interest. Presently, there are a number of different strategies which have been proposed to accomplish this task, but these have not been systematically compared. One of the most intensively analyzed regions of the human genome is band 4p16.3, site of the recently cloned Huntington's disease gene. Detailed genetic and physical maps are available, YAC and cosmid contigs have been constructed, and complete DNA sequence of a 2Mb segment is currently being generated. We propose to use this region to perform a much-needed assessment of strategies for saturating a genomic region with gene sequences and for detailed comparison of the homologous region in the mouse. We will compare exon amplification, cDNA selection based on affinity labeling, and sequence-based exon prediction as means of isolating expressed sequences, while thereby saturating this segment of 4p16.3 with genes. The equivalent region of the mouse genome will also be cloned and the content, orientation and genomic coverage of genes will be compared with man. This study should provide valuable experience for applying saturation strategies to any specified genomic region for the isolation of disease genes, for facilitating more detailed mapping strategies, or for making structural associations within or between species.