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Construction of medaka BAC library

青鳉BAC文库的构建

基本信息

批准号：
11236209
负责人：
ASAKAWA Shuichi
金额：
$ 29.25万
依托单位：
Keio University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research on Priority Areas
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2003
项目状态：
已结题

项目摘要

1. We have constructed a set of high-quality Medaka genome BAC library from the inbred HNI strain. About 96,000 clones with average insert size of 160 kb cover 20 times the medaka genome. These clones were individually stocked in 250 of 384-well plates. These clones were also spotted on nylon membranes for colony hybridization screening.2. We have established a novel procedure to efficiently obtain BAC-end sequences, in which the BAC-DNAs are prepared with 96-well format using Qiagen R.E.A.L. Prep 96 and are sequenced directly by ABI3700/3730 capillary sequencers. We have finished the end-sequencing of 11, 904 Hd-rR BAC clones, 8,160 HNI BAC clones, and 1,152 Cab BAC clones. The total 42,432 end sequence data were deposited in our database as GSS (Genome survey sequence).3. We also established the two step four dimensional (4D-)PCR screening system for the Hd-rR BAC library. First screening is against superpools each consisting of 1,536 BAC clones. Then, second screening is against the positive superpools identified by the first screening. Individual clones of each superpool are addressed 4-dimensionally, therefore we can identify single clone from the l,536 clones by PCR reactions for 28 second pools. We have prepared twenty superpools, therefore total 30,720 clones are accessible which correspond to 8 times of Medaka genome. A set of screening can be finished within a day.4. We started entire sequencing of the medaka LG22 chromosome (about 20 Mb) in collaboration with several research groups in the MGI. We have covered 11Mb of genomic region with BAC contigs and have finished sequencing 7Mb of the chromosome.5. To date, we provided many BAC clones, materials for screenings, and BAC-end sequence data to several research groups. It is noted that the BAC libraries have contributed positional cloning of several genes including medaka sex determining gene and analysis of gene structure as well as constructions of genome-wide BAC contig map and linkage map.

1.我们构建了一套高质量的青鳉基因组BAC文库。约96，000个平均插入片段大小为160 kb的克隆覆盖青鳉基因组的20倍。将这些克隆分别储存在250个384孔板中。将这些克隆点在尼龙膜上进行菌落杂交筛选.我们已经建立了一种新的程序，以有效地获得BAC-末端序列，其中BAC-DNA制备与96孔格式使用Qiagen R. E. A. L. Prep 96和直接测序ABI 3700/3730毛细管测序仪。我们已经完成了11，904个Hd-rR BAC克隆、8，160个HNI BAC克隆和1，152个Cab BAC克隆的末端测序。共获得42，432条末端序列数据，作为GSS（Genome survey sequence）数据库.我们还建立了Hd-rRBAC文库的两步四维PCR筛选体系。第一次筛选是针对每个由1，536个BAC克隆组成的超级库。然后，第二次筛选是针对通过第一次筛选鉴定的阳性超级池。每个超级库的单个克隆被4维寻址，因此我们可以通过28秒库的PCR反应从1，536个克隆中鉴定单个克隆。我们已经准备了20个超级池，因此总共有30，720个克隆是可访问的，对应于青鳉基因组的8倍。一套筛选可在一天内完成。4.我们与MGI的几个研究小组合作，开始了青鳉LG 22染色体（约20 Mb）的完整测序。我们已经用BAC重叠群覆盖了11 Mb的基因组区域，并完成了7 Mb的染色体测序。到目前为止，我们提供了许多BAC克隆，筛选材料，和BAC末端序列数据的几个研究小组。值得注意的是，BAC文库为青鳉性别决定基因等多个基因的定位克隆和基因结构分析以及全基因组BAC重叠群图谱和连锁图谱的构建做出了贡献。

项目成果

期刊论文数量（74）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Matsuda M, Nagahama Y, Shinomiya A, Sato T, Matsuda C, Kobayashi T, Morrey CE, Shibata N, Asakawa S, Shimizu N, Hori H, Hamaguchi S, Sakaizumi M.: "DMY is a Y-specific DM-domain gene required for male development in the medaka fish"Nature. 417. 559-563 (2

Matsuda M、Nagahama Y、Shinomiya A、Sato T、Matsuda C、Kobayashi T、Morrey CE、Shibata N、Asakawa S、Shimizu N、Hori H、Hamaguchi S、Sakaizumi M.：“DMY 是 Y 特异性 DM 结构域