A novel method for making nested deletions and its application for sequencing of a 300 kb region of human APP locus

Masahira Hattori*, Fujiko Tsukahara, Yoshiaki Furuhata, Hiroshi Tanahashi, Matsumi Hirose, Masae Saito, Shiho Tsukuni, Yoshiyuki Sakaki

*この研究の対応する著者

研究成果: Article査読

62 被引用数 (Scopus)

抄録

We developed a novel in vitro method for making nested deletions and applied it to a large-scale DNA sequencing. A DNA fragment to be sequenced (up to 15 kb long) was cloned with a new vector possessing two unique Sfil sites, digested by Sfil and ligated to generate a large head-to-tail concatemer. The large concatemer was randomly fragmented by sonication and then redigested by Sfil to separate insert and vector DNAs. The fragments of various length were then cloned into the other vector(s) specifically designed for selective cloning of insert-derived DNA fragments to generate a library of nested deletions. This method allowed a single person to generate > 20 nested deletion libraries sufficient to cover 100 kb in a few days. We applied the method for sequencing of P1 clones and successfully determined the complete sequence-of ≃300 kb of the human amyloid precursor protein (APP) locus on chromosome 21 with a redundancy of 3.8, reasonably low cost and very few gaps remaining to be closed. Development of some new instruments and software is also described which makes this method more applicable for large-scale sequencing.

本文言語English
ページ(範囲)1802-1808
ページ数7
ジャーナルNucleic Acids Research
25
9
DOI
出版ステータスPublished - 1997 5月 1
外部発表はい

ASJC Scopus subject areas

  • 遺伝学

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