1995 Fiscal Year Final Research Report Summary
"Development of a method for accurate determination of nucleotide sequences using a DNA sequencer"
| Project/Area Number |
06558103
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| Research Category |
Grant-in-Aid for Developmental Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Molecular biology
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| Research Institution | Kobe University |
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Principal Investigator |
ISONO Katsumi Kobe Univ., Faculty of Science Professor, 理学部, 教授 (70011640)
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| Co-Investigator(Kenkyū-buntansha) |
SAKURAI Toshiyuki Hitachi Electronic Engineering, Engineer, 技術本部研究部, 技師
HONJOU Atsuko Kobe Univ., Faculty of Science, Assistant, 理学部, 助手 (30229249)
KITAKAWA Madoka Kobe Univ., Faculty of Science, Assistant, 理学部, 助手 (70169853)
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| Project Period (FY) |
1994 – 1995
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| Keywords | Transposon / PCR / Magnetic beads / Nucleotide sequencing / Lambda phage / Base-caller / Control sequence database |
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| Research Abstract |
This project was aimed at establishing a method for more accurate and reliable base-calling system for the determination of nucleotide sequences using a DNA sequencer from Hitachi, model SQ3000. For this purpose, we obtained a large amount of nucleotide sequence raw data according to the transposon-facilitated PCR-based sequencing system of Kasai et al. (1992) using magnetic beads. As the template for analysis, we chose lambda phage DNA because of its easiness for preparation. We analyzed the distribution of peak distances and its dependence on the local nucleotide sequences and compared the sequence data obtained between electrophoresis lanes. The data were organized as a database and used for the probability calculation of individual nucleotides in the base-calling at each position of the sequence data. It thus turned out that the modified base-caller was more accurate in determining the sequence than the previous one : the accuracy level was at 97-100 % up to 300 bases and at 94-98 % up to 400 bases. Above 400 bases, however, the accuracy level was not very much improved by the new base-caller and it was found that other factors such as the quality of samples and acrylamide gel affected more on the base-calling accuracy.
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[Publications] Berg, C.M., Wang, G., Isono, K., Kasai, H., Berg, D.E.: "Transposon-facilitated large-scale DNA sequencing. In "Automated Sequencing and Analysis"" Adams, M.D.et al.eds., Academic Press. 51-59 (1994)
Description
「研究成果報告書概要(欧文)」より
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[Publications] Kasai, H., Kim, S.-O., Isono, S., Borodovsky, M., Isono, K.: "Solid Phase Nucleotide Sequencing and Its Application to Genome Analysis of Escherichia coli K-12. In "Advances in Biomagnetic Separation" Uhlen, M.et al.eds., Eaton Publishing. 113-125 (1994)
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「研究成果報告書概要(欧文)」より
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[Publications] Kasai, H., Kim, S.-O., Isono, S., Moriya, H., Honjo, A., and Isono, K.: "Use of trans-poson, PCR and magnetic beads for the genome analysis of E.coli K-12 : Further improvements of the method and analysis of the terC region. Proceedings of JBA Symposium on Microorganism DNA" 37-46 (1994)
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[Publications] Hirosawa, M., Kaneko, T., Tabata, S., McIninch, J.D., Hays, W.S., Borodovsky, M., Isono, K.: "Computer survey for likely genes in the one megabase contiguous genomic sequence data of of Synechocystis sp.strain PCC6803." DNA Res.2. 239-246 (1995)
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「研究成果報告書概要(欧文)」より
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