| Project/Area Number |
06454661
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Biophysics
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| Research Institution | Saitama University |
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Principal Investigator |
NISHIGAKI Koichi Saitama Univ., Fac.Eng., Asso.Professor, 工学部, 助教授 (10107378)
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| Co-Investigator(Kenkyū-buntansha) |
SUZUKI Miho Saitama Univ., Fac.Eng., Assi.Professor, 工学部, 助手 (60222064)
SUEMITSU Takashi Saitama Univ., Fac.Scl., Asso, Professor, 理学部, 助教授 (40092019)
HUSIMI Yuzuru Saitama Univ., Fac.Eng., Professor, 工学部, 教授 (80011641)
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| Project Period (FY) |
1994 – 1995
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| Project Status |
Completed (Fiscal Year 1995)
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| Budget Amount *help |
¥6,400,000 (Direct Cost: ¥6,400,000)
Fiscal Year 1995: ¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1994: ¥4,400,000 (Direct Cost: ¥4,400,000)
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| Keywords | ROSOR method / Rondom PCR / Tetramer Library / Enzymatic Block Synthesis / Human Genome Project / Base Sequencing / PCR Relay / Planar Representation of Sequence / 酵素的ブロック合成 / 配列平面表示法 / 酵素合成 / ダイレクトシーケンシング / オリゴヌクレオチド |
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| Research Abstract |
1.Rapid synthesis of oligonucleotides We have explored the way to make oligonucleotide (19mer) which are sufficient to be used for PCR applied to human genome. In order to settle the problem related to purity and yields of the PCR products, we introduced a semi-solid phase synthesis in which substrate oligonucleotides are free in solution when they are extended while they are bound to a bead when unreacted reagents are washed out. Some results were obtained as to this.
2.Random PCR Preparing template DNAs from various organisms, we performed more than 100 combinations of genome profilings which include a random PCR process. Through these experiments, we can obtain confirmation that random PCR can be widely used for the purpose of setting anchoring sites on a genome DNA.We also reached to a new concept that anchoring by random PCR can play the major role in genome sequencing since it needs a limited number of oligonucleotides (Random PCR-based fast ROSOR method).
3.Demonstration of PCR relay Using E.coli genome DNA (4.7Mb), a library of restriction enzyme Sau3AI partial digestion products was contructed. Specific PCR was successfully carried out with this library, generating the aimed DNA fragments. Thus, overall scheme of PCR relay was demonstrated by this result.
4.Related techniques developed PCR in a tiny aliquot (0.01mul) was possible. Multi-microcells (30 cells/cm2) were formed out of polyacrylamide gel and were usable for PCR reactions. Computer programs to help analyze voluminous sequence data were developed (PRS ; Planar representation of sequence)and were shown to be useful for sequence data analyzes.
In conclusion, physically-non-dividing strategy, ROSOR,to sequence voluminous genome DNA was demonstrated to be quite feasible, and, in addition, random PCR based-sequencing proved to be highly effective.
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