A Novel Method for Generating Nested Deletions Using the in Vitro Bacteriophage T3 DNA Packaging System |
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Authors: | Kawarabayasi Yutaka; Tanaka Ayako; Ohara Osamu; Arakawa Taku; Oka Masanori; Kato Hisako; Morita Miyo; Fujisawa Hisao |
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Institution: | 1Kazusa DNA Research Institute 1532-3 Yana-uchino, Kisarazu, Chiba 292, Japan
2Tsuruga Institute of Biotechnology, Toyobo Co., Ltd. Tsuruga, Fukui 914, Japan
3Laboratory of Natural Science, Ohtani University Kita-ku, Kyoto 603, Japan
4Department of Botany, Faculty of Science, Kyoto University Sakyo-ku, Kyoto 606, Japan |
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Abstract: | To sequence a DNA segment inserted into a cosmid vector underthe directed sequencing strategy, we established a simple andrapid method for generating nested deletions which uses thein vitro packaging system of bacteriophage T3 DNA. The principleis based on the previous finding that this system can translocateany linear double-stranded DNA up to 40 kb into the phage capsidin a time-dependent manner and the encapsulated DNA becomesDNase-resistant. For this purpose, we constructed a cosmid vectorthat carries two different antibiotic selection markers at bothsides of the multiple cloning site, and after insertion of aDNA segment, the clone was linearized by -terminase at the cossite. After the packaging reaction in vitro followed by DNasetreatment, the encapsulated DNA was introduced into Escherichiacoli cells to give clones with unidirectional deletions by differentialantibiotic selection. Restriction and sequence analyses of deletionclones demonstrated that an ordered set of clones with nesteddeletions, ranging from less than 1 kb to 25 kb, was createdfrom either the end of the DNA segment. Thus, nested deletionclones that cover the entire region of a 40-kb cosmid insertcan be obtained by a single packaging reaction, and its restrictionmap can be simultaneously obtained. |
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Keywords: | T3 bacteriophage in vitro DNA packaging nested deletion DNA sequencing cosmid |
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