Construction of Ordered-Clones Library for Human Chromosome 21

本项目旨在利用酵母人工染色体（YAG）系统构建人类21号染色体有序克隆文库。从只含21号染色体的人与中国仓鼠杂交细胞中分离DNA，构建了第一个YAG文库。然而，由于很难确定21号染色体是否在杂交细胞中保留为正常染色体，并且由于新的快速筛选YAC文库的方法已经开发出来，因此没有必要将杂交细胞作为DNA来源-因此，从核型正常的细胞中分离出人类DNA构建第二文库。由于大yag有利于筛选染色体特异性克隆和促进克隆顺序的确定，因此提出了改进的大yag克隆方法。human-YAG库包含大约14000个克隆，平均大小为380kb。据估计，该文库中YACs的总长度相当于人类单倍体基因组长度的1.7倍。采用PCR筛选方法分离得到9个21号染色体特异性克隆，并构建了限制性内切图谱。末端特异性dna片段的分离和重叠克隆的筛选正在进行中。到目前为止，覆盖整个21号染色体的有序克隆还没有被分离出来。但是，由于已经建立了包含大型克隆的YAC文库，并且引入了快速灵敏的筛选方法，因此，有序克隆文库的建设应在不久的将来进行。

The aim of this project is construction of ordered-clones library for human chromosome 21 using the large insert cloning system, yeast artificial-chromosomes (YAG) system.1. The first YAG library was constructed with DNA isolated from the human X Chinese hamster hybrid cells which contained chromosome 21 as only human chromosome. However, because it seemed to be difficult to confirm whether the chromosome 21 was kept as normal chromosome in the hybrid cells or not, and because the new rapid-method for screening YAC library has developed, it was not necessary to use the hybrid cells as source of DNA- Therefore, the second library was constructed with human DNA isolated from the cells whose karyotype was normal.2. The improved method for cloning large YACs was developed because the large YAGs would facilitate the screening for chromosome-specific clones and promote to determine the order of the clones.3. The human-YAG library contained about 14,000 clones whose average size was 380 kb. It was estimated that the total length of YACs in the library was equivalent to 1.7-fold of the length of the human haploid genome.4. Nine chromosome 21-specific clones were isolated using PCR based screening method and these restriction maps were constructed.5. Isolation of the end specific DNA-fragments and screening of the overlapped clones are in progress.Until now, the set of ordered clones which cover whole chromosome 21 have still not isolated. However, the ordered clones library should be constructed in the near future because the YAC library containing large clones has been built and because the rapid and sensitive screening method has been introduced.