PFGE and YAC analysis of the Arabidopsis genome

Procedures for the preparation, analysis, and cloning of large DNA molecules from Arabidopsis thaliana are described. Root callus protoplasts are used to prepare large DNA molecules in agarose plugs or in liquid. Large DNA preparations can be used both for restriction enzyme analysis of the Arabidopsis genome and for molecular cloning of large segments of DNA into yeast using artificial chromosome vectors. Size selection of ligation products from pulsed-field gels has allowed for the cloning of YACs with an average insert size of 0.25 Mbp; yeast artificial chromosomes range in size from 0.07 to 0.7 Mbp. PFGE and blot hybridization analyses of Arabidopsis DNA-containing yeast artificial chromosomes demonstrated that both unique and highly repeated DNA sequences are represented. The roles of PFGE and yeast artificial chromosome cloning methods for physical mapping of the Arabidopsis genome are discussed.