Establishment of framework P1 clones for map-based cloning and genome sequencing: Direct RFLP mapping of large clones

Daisuke Shibata, Motoaki Seki, Norihiro Mitsukawa, Nobuaki Hayashida, Takuya Ito, Teruaki Taji, Tomohiko Tsuge, Minami Matsui, Toyoaki Anai, Yao Guang Liu, Robert F. Whittier, Kazuo Shinozaki

Research output: Contribution to journalArticlepeer-review

Abstract

Large insert capacity, clone stability and convenient propagation in Escherichia coli have made bacterial artificial chromosome and phage P1 vector-based libraries the first choice for large-scale sequencing projects, and these libraries have also proven useful for chromosome walking. The application of these libraries for either purpose is greatly facilitated by the establishment of a set of framework clones distributed across the genome. Using a P1-based library of Arabidopsis thaliana with genomic inserts of 70- 90 kb (Liu, Y.-G., Mitsukawa, N., Vazquez-Tello, A., Whittier, R.F., 1995. Generation of a high-quality P1 library of Arabidopsis suitable for chromosome walking. Plant J. 7, 351-358), we have now established such a set of framework clones. To date, such clones have usually been identified by hybridization to smaller, previously mapped clones that detect restriction fragment length polymorphisms (RFLPs). In order to establish framework clones more efficiently, we refined protocols for P1 clone DNA isolation and RFLP detection in order to employ whole P1 clones directly as probes. This strategy enabled a very high rate of RFLP detection, and obviated the need to screen the P1 library with smaller RFLP probes. Altogether 95 clones were mapped providing a framework into which further clones can be integrated by physical overlap.

Original languageEnglish
Pages (from-to)31-38
Number of pages8
JournalGene
Volume225
Issue number1-2
DOIs
Publication statusPublished - Dec 28 1998
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Genetics

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