Construction of Ordered-Clones Library for Human Chromosome 21

人21号染色体有序克隆文库的构建

• 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
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-63571082/
• 关键词: Human chromosome 21; YAC; Ordered-clones library; 酵母人工染色体ベクター; コンティグマップ; アガロース顆粒法; Alu配列; ヒト、ハムスター雑種細胞; 長鎖DNA

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--酵母人工染色体系统，构建人21号染色体的有序克隆文库。从含有21号染色体的人X中国仓鼠杂交细胞中提取DNA，构建了第一个以人为唯一染色体的中国仓鼠杂交细胞基因组DNA文库。但由于难以确定21号染色体在杂交细胞中是否作为正常染色体存在，以及YAC文库快速筛选新方法的发展，不再需要将杂交细胞作为DNA来源，因此，从核型正常的细胞中提取人DNA，构建了第二个文库。大YAC克隆方法的改进是因为大的YAC有利于染色体特异性克隆的筛选和克隆顺序的确定。该文库包含约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。采用基于聚合酶链式反应的筛选方法，筛选出9个21号染色体特异克隆，构建了相应的限制性内切酶图谱。末端特异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集还没有分离到。但由于YAC文库的构建和快速、灵敏的筛选方法的引入，YAC大克隆文库的构建是近期需要解决的问题。

项目成果

Takashi Imai: "Second-Generation Approach to the Construction of Yeast Artificial-Chromosome Libraries"

Takashi Imai：“酵母人工染色体文库构建的第二代方法”

今井高志、添田栄一: "Bio medica3巻10号" 医薬出版社, 1041-1045 (1988)

Takashi Imai、Eiichi Soeda：《Bio medica Volume 3 No. 10》Iyaku Shuppansha，1041-1045 (1988)

Takashi Imai and Maynard Olson: "Second-Generation Approach to the Construction of Yeast Artificial-Chromosome Libraries"

Takashi Imai 和 Maynard Olson：“酵母人工染色体文库构建的第二代方法”

Takashi Imai: "SecondーGeneration Approach to the Construction of Yeast ArtificialーChromosome Libraries"

Takashi Imai：“酵母人工染色体文库构建的第二代方法”