| Project/Area Number | 07558295 |
|---|
| Research Category |
Grant-in-Aid for Scientific Research (A)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 展開研究 |
|---|
| Research Field |
Biomedical engineering/Biological material science
|
|---|
| Research Institution | The Institute of Physical and Chemical Research (RIKEN)(1996-1997)
Okayama University(1995) |
|---|
Principal Investigator |
SOEDA Eiichi(1996-1997) RIKEN,Gene Bank, Senior Researcher, ジーンバンク室, 副主任研究員 (00039330)
片山 泰人(1995) 岡山大学, 医学部, 講師 (40033352)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
HOSOKAWA Keiichi Kawasaki Medical School, Professor, 医学部, 教授 (00104787)
KATAYAMA Yasuhito Medical School of Okayama, Lecturer, 医学部, 講師 (40033352)
添田 栄一 理化学研究所, ジーンバンク室, 副主任研究員 (00039330)
|
|---|
| Project Period (FY) |
1995 – 1997
|
| Project Status |
Completed(Fiscal Year 1997)
|
| Budget Amount *help |
¥5,500,000 (Direct Cost : ¥5,500,000)
Fiscal Year 1997 : ¥2,600,000 (Direct Cost : ¥2,600,000)
Fiscal Year 1996 : ¥2,900,000 (Direct Cost : ¥2,900,000)
|
|---|
| Keywords | sequence-ready map / CpG-islands / chromosome walking / CpG-tagged sequence / PAC contigs / 筋萎縮側鎖硬化症 / PAC / ダウン症 / PCRスクリーニング / 大腸菌人工染色体 / ショットガンシークエンス / 染色体21番疾病関連遺伝子 / ゲノム物理地図 / コスミド整列化 / CpGアイランド / PAC末端シークエンス / ヒト21番染色体 / 遺伝子物理地図 / 遺伝子発現地図 / ヒトゲノム解析計画 / ALS |
|---|
| Research Abstract |
A sequence-ready map or the human genome will be generated from PAC contigs because they have provided unique materials for high resolution map and sequencing. We have developed rapid screening and chromosome walking with which almost all gaps were able to be sealed with use of a half million PAC clones, representing approx. 20-fold equivalents of the total genome.
We extracted 1440 DNAs from 384-well plates which have been arrayd with PAC clones, and prepared "PCR screening kits" for the plate selection. For the isolation of clone from the plate, we devised "ditch plates" for mixing bacteria for PCR with which enabled us to isolate any PAC with STS or PCR-primers within a couple of days. For chromosome walking, we applied direct sequencing from the ends of PAC inserts and read consistently more than 500 bases from which we designed PCR-primers for the confirmation of the existing contigs and screening of next PACs. With this system, we have completed a 7-Mb high resolution map from SOD
… More
l to ETS2. The full integrity of PAC and high dense cosmid overlap map shows the fidelity in cloning with the bacterial system.
Approximately 60 % of the expressed genes of the human genome is known to be associated with CpG islands, most of which are able to be detected with BssHII, EagI and SacII.To help isolate and identify genes, we have sought CpG-islands in the cosmid/PAC contigs with these diagnostic enzymes, assuming close clustering (within l kb) of two or more cutting sites as potential CpG islands. The sequences around the sites have revealed known and unknown genes in this region. Of those, the sequences in PAC 25P16 clone identified.CBR and its analogue, respectively. To verify this approach, we completed the sequence of this clone in shotgun strategies and defined seven CpG islands with which three known and three unknown genes were associated. This approach, designated as CpG-tagged sequence, is rapid and convenient for searching genes and will provide a scaffold for the construction of a transcription map. Less
|
