PMID: 8580969Jan 1, 1996Paper

An improved method of partially digesting plant megabase DNA suitable for YAC cloning: application to the construction of a 5.5 genome equivalent YAC library of tomato

The Plant Journal : for Cell and Molecular Biology
G BonnemaP Zabel

Abstract

An improved method for preparing partially digested tomato DNA has been developed, that is suitable for YAC cloning. It involves (i) isolation of high molecular-weight DNA from agarose-embedded leaf protoplasts, (ii) controlled partial digestion in situ using EcoRl endonuclease in the presence of EcoRI methylase (M. EcoRl), and (iii) fractionation of the partial digest on a Clamped Homogeneous Electric Fields (CHEF) gel. Unlike methods commonly used for generating partial digests, the present method allows one to produce digests in which the bulk of restriction fragments are of the desired size. Use of these partial digests in constructing YAC libraries of the tomato lines Moneymaker-Cf4 and VFNT Cherry resulted in libraries (total 21,060 clones, 5.5 genome equivalents) in which 80% of the YACs have inserts between 200 and 600 kb. Both libraries have been screened with selected RFLP markers linked to the Cladosporium fulvum Cf4 locus on chromosome 1, using a three-dimensional PCR-based screening technique. To this end, the RFLP markers have been sequenced to allow for the synthesis of specific primers. Thus, for each marker tested several YAC clones have been isolated, including a family of clones that carry leucine-rich repeat...Continue Reading

Citations

Jan 5, 2012·Critical Reviews in Food Science and Nutrition·Andrés Cruz-Hernández, Octavio Paredes-López
May 5, 2001·Annual Review of Plant Physiology and Plant Molecular Biology·Jim Giovannoni
Mar 28, 2008·International Journal of Plant Genomics·Majid R Foolad
Apr 26, 2000·Proceedings of the National Academy of Sciences of the United States of America·B F BrandwagtH J Nijkamp
Feb 17, 1999·Proceedings of the National Academy of Sciences of the United States of America·P MartinezE H Davidson
Jan 1, 1997·TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik·A van TuinenP Zabel
Mar 26, 2009·Critical Reviews in Biotechnology·Vikrant GuptaArun K Sharma
Feb 14, 2004·TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik·S SeahV M Williamson
Sep 12, 2002·Annual Review of Plant Biology·Didier G Schaefer

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