CONSTRUCTION OF A PHYSICAL MAP OF HUMAN CHROMOSOME 21

这个项目的总体目标是生成一个完整的物理地图 至少是人类21号染色体的长臂。这条染色体是 对几种人类遗传性疾病很重要，包括唐氏综合症， 阿尔茨海默病、肌萎缩侧索硬化症、进行性肌阵挛 癫痫，可能还有白血病。 一组酵母人工染色体(YAC)，覆盖约20%的 21号染色体(21q)的长臂已经在这里产生 实验室代表21号染色体联合YAC进行筛查工作。 此外，本实验室还构建了2LQ的脉冲场凝胶图谱 跨越4400万个碱基对的DNA，或超过90%的估计 这个染色体臂的大小。可用于构建地图的试剂，在 一组跨越染色体的有序的、重叠的YAC的形式 包括3个不同YAC库，覆盖范围约为13倍 在人类基因组中，107个额外的区域定位的染色体 21-特异性DNA探针尚未用于筛选YAC文库，非常 详细的中国仓鼠/人类细胞杂交区域定位小组 本实验室产生并鉴定了21号染色体和2号染色体 来自显微切割的21S染色体的微克隆文库，包括AT 至少740,000个21号染色体克隆。要使用的现成方法包括 基于PCR和杂交的YAC文库筛选方案，YAC完 利用Vectorette和Alu载体进行克隆，长距离限制 YAC的定位和荧光原位杂交(FISH) 中期染色体以YAC为探针评价YAC嵌合体。一个 新技术RecA辅助限制性内切酶(Rare)切割， 将被评估以帮助验证YAC重叠群的保真度， 填补空白(闭合)，以及用于其他测绘目的。

The overall goal of this project is to generate a complete physical map of at least the long arm of human chromosome 21. This chromosome is important for several human genetic diseases, including Down syndrome, Alzheimer disease, amyotrophic lateral sclerosis, progressive myoclonus epilepsy, and probably leukemia. A set of yeast artificial chromosomes (YACs) covering about 20% of the long arm of chromosome 21 (21q) already has been generated in this laboratory on behalf of the chromosome 21 Joint YAC Screening Effort. Moreover, this laboratory has constructed a pulsed field gel map of 2lq spanning over 44 million base pairs of DNA, or over 90% of the estimated size of this chromosome arm. Reagents available to construct the map, in the form of a set of ordered, overlapping YACs spanning the chromosome include 3 different YAC libraries representing roughly 13-fold coverage of the human genome, 107 additional regionally mapped chromosome 21-specific DNA probes not yet used to screen YAC libraries, a very detailed Chinese hamster/human cell hybrid regional mapping panel for chromosome 21 generated and characterized in this laboratory and 2 microclone libraries from microdissected chromosome 21s comprising at least 740,000 chromosome 21 clones. Methods in place to be used include both PCR and hybridization based YAC library screening protocols, YAC end cloning by Vectorette PCR and alu-vector PCR, long-range restriction mapping of YACs, and fluorescence in situ hybridization (FISH) to metaphase chromosomes using YACs as probes to assess YAC chimerism. A new technique, RecA Assisted Restriction Endonuclease (RARE) Cleavage, will be assessed to aid in validating the fidelity of the YAC contig, filling in gaps (closure), and for other mapping purposes.