A novel method for making nested deletions and its application for sequencing of a 300 kb region of human APP locus
| Autor: | Hiroshi Tanahashi, Masae Saito, Yoshiyuki Sakaki, Yoshiaki Furuhata, Masahira Hattori, Fujiko Tsukahara, Shiho Tsukuni, Matsumi Hirose |
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| Jazyk: | angličtina |
| Rok vydání: | 1997 |
| Předmět: |
Genetics
Cloning DNA Complementary Base Sequence Concatemer Chromosomes Human Pair 21 Molecular Sequence Data Chromosome Mapping Locus (genetics) Biology DNA sequencing Complete sequence chemistry.chemical_compound Amyloid beta-Protein Precursor chemistry Humans A-DNA Chromosome 21 DNA Research Article Sequence Deletion |
| Popis: | 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 Sfi I sites, digested by Sfi I and ligated to generate a large head-to-tail concatemer. The large concatemer was randomly fragmented by sonication and then redigested by Sfi I 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 approximately 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. |
| Databáze: | OpenAIRE |
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