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

Large insert capacity, clone stability and convenient propagation in Escher ichia coli have made bacterial artificial chromosome and phage P1 vector-ba sed libraries the first choice for large-scale sequencing projects, and the se libraries have also proven useful for chromosome walking. The applicatio n of these libraries for either purpose is greatly facilitated by the estab lishment of a set of framework clones distributed across the genome. Using a Pi-based library of Arabidopsis thaliana with genomic inserts of 70-90 kb (Liu, Y.-G., Mitsukawa, N., Vazquez-Tello, A., Whittler, R.F., 1995. Gener ation of a high-quality P1 library of Arabidopsis suitable for chromosome w alking. Plant J. 7, 351-358), we have now established such a set of framewo rk clones. To date, such clones have usually been identified by hybridizati on to smaller, previously mapped clones that detect restriction fragment le ngth polymorphisms (RFLPs). In order to establish framework clones more eff iciently, we refined protocols for PI clone DNA isolation and RFLP detectio n in order to employ whole P1 clones directly as probes. This strategy enab led 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 prov iding a framework into which further clones can be integrated by physical o verlap. (C) 1998 Elsevier Science B.V. All rights reserved.