Construction of Ordered-Clones Library for Human Chromosome 21

人21号染色体有序克隆文库的构建

基本信息

批准号：
63571082
负责人：
IMAI Takashi
金额：
$ 1.34万
依托单位：
The Institute of Physical and Chemical Research
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1988
资助国家：
日本
起止时间：
1988 至 1989
项目状态：
已结题

项目摘要

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.

本项目的目的是利用大插入克隆系统——酵母人工染色体（YAG）系统构建人类21号染色体的有序克隆文库。 1．第一个YAG文库是用从人X中国仓鼠杂交细胞中分离的DNA构建的，该细胞仅含有21号染色体作为人类染色体。然而，由于似乎很难确认杂交细胞中21号染色体是否保留为正常染色体，并且由于筛选YAC文库的新快速方法已经开发出来，因此没有必要使用杂交细胞作为DNA来源-因此，用核型正常的细胞中分离的人类DNA构建了第二个文库。 2．由于大YAGs有利于筛选染色体特异性克隆，并有助于确定克隆的顺序，因此提出了改进的大YACs克隆方法。 3．人类 YAG 文库包含约 14,000 个克隆，平均大小为 380 kb。据估计，文库中YAC的总长度相当于人类单倍体基因组长度的1.7倍。4．采用PCR筛选方法分离得到9个21号染色体特异性克隆，并构建了酶切图谱。 5．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组仍未分离。然而，由于含有大克隆的YAC文库已经建立，并且引入了快速灵敏的筛选方法，有序克隆文库应该在不久的将来构建。