Development of large DNA methods for plants: molecular cloning of large segments of Arabidopsis and carrot DNA into yeast

Abstract

Procedures for the preparation, analysis and cloning of large DNA molecules from two different plant species are described. Arabidopsis and carrot protoplasts were used for the preparation of large DNA molecules in agarose "plugs" or in solution. Pulsed-field gel electrophoresis (PFGE) analysis of large plant DNA preparations using a contour-clamped homogeneous field (CHEF) apparatus indicated that the size of the DNA was at least 12 Mb. Large DNA preparations were shown to be useful for restriction enzyme analysis of the Arabidopsis genome using both frequent and infrequent cutting enzymes and for the molecular cloning of large segments of DNA into yeast using artificial chromosome (YAC) vectors. PFGE and blot hybridization analysis of Arabidopsis and carrot DNA-containing YACs indicated that both unique and highly repeated DNA sequences were represented in these libraries.