| Co-Investigator(Kenkyū-buntansha) |
URANO Gen Aloka Co. Ltd. Researcher, 研究所, 研究員
OHARA Meguru Kobe Univ., Laboratory of Molecular Biology Assistant, 遺伝子実験施設, 助手 (00204272)
TACHIBANA Hideki Kobe Univ., Faculty of Science Assoc. Prof., 理学部, 助教授 (70126118)
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| Research Abstract |
To make the "genome analysis" of organisms such as Escherichia coli and Saccharomyces cerevisiae possible, it is necessary to develope methods for more rapid and efficient determination of nucleotide sequences. Since the most labor-and time-consuming step in nucleotide sequencing is the subcloning of truncated fragments, an alternative method of sequencing without subcloning was sought. An "ordered clone bank" was established in E. coli by Kohara et al. (1987) and partly in S. cerevisiae by Yoshikawa and Isono (1990 ; 1991). Therefore, if there is a method of amplifying and sequencing a desired region of a clone in the bank, then a rapid and more efficient genome sequencing will be achieved. For this purpose, the "mini-transposon" constructed by Phadnis et al. (1990) appeared to be ideal when used in conjunction with a dnaB (amber) mutant strain termed DK-21 for selection. Mini-transposons were accordingly introduced into individual E. coli and S. cerevisiae ordered clones and the resultant derivatives were analyzed with respect to their positions of transposon insertion. The region flanked by a mini-transposon inserted into the chromosomal segment in a clone and either the right or left arm of the phage vector was then amplified by PCR and the amplified fragment was subjected to sequencing using a DNA sequencer. Thus, it has become possible to obtain nucleotide sequence data without performing subclonings. Furthermore, to facilitate faster and more accurate assembly of the nucleotide sequence data thus obtained, a method was developed in which a control sample of already known sequence such as M13 phage DNA was run in parallel and was used to position each nucleotide in the sequence data correctly.
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