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.

该项目的目的是使用大型插入克隆系统，酵母人工染色体（YAG）系统来构建用于人类染色体的有序clones库。1。第一个YAG文库是由从人类X中国仓鼠杂种细胞中分离出的DNA，其中包含21染色体作为人类染色体。但是，由于似乎很难确认21号染色体在混合细胞中是否保持正常染色体，并且由于新的筛选YAC库的新快速方法已经开发出来，因此不必将混合细胞用作DNA的来源 - 因此，第二个库是与karyotype隔离的第二个库构建的第二库。开发了克隆大YAC的改进方法，因为大型YAG会促进染色体特异性克隆的筛选，并促进确定克隆的顺序。3。 Human-Yag图书馆包含大约14,000个克隆，其平均大小为380 KB。据估计，文库中YAC的总长度相当于人单倍体基因组长度的1.7倍。4。使用基于PCR的筛选方法分离了九个染色体21特异性克隆，并构建了这些限制图5。末端特异性DNA碎片的隔离和重叠克隆的筛选正在进行中。直到现在，覆盖整个染色体21的有序克隆仍未隔离。但是，由于已经构建了包含大型克隆的YAC库，并且已经引入了快速敏感的筛选方法，因此应在不久的将来构建有序的克隆库。

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”