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

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61 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)1802-1808
Number of pages7
JournalNucleic Acids Research
Volume25
Issue number9
DOIs
Publication statusPublished - 1997 May 1
Externally publishedYes

ASJC Scopus subject areas

  • Genetics

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    Hattori, M., Tsukahara, F., Furuhata, Y., Tanahashi, H., Hirose, M., Saito, M., Tsukuni, S., & Sakaki, Y. (1997). A novel method for making nested deletions and its application for sequencing of a 300 kb region of human APP locus. Nucleic Acids Research, 25(9), 1802-1808. https://doi.org/10.1093/nar/25.9.1802