Plant & Animal Genome VII Conference

P19

PHYSICAL MAPPING OF BACTERIAL ARTIFICIAL CHROMOSOME CLONES USING FLUORESCENCE IN SITU HYBRIDIZATION

University of Wisconsin-Madison, Department of Horticulture, 1575 Linden Drive, Madison, WI 53705 USA

Physical mapping of large genomic DNA clones generally proceeds by subcloning, restriction mapping and sequencing. We developed a physical mapping technique on individual bacterial artificial chromosome (BAC) molecules using fluorescence in situ hybridization (FISH). The BAC inserts or intact circular BAC molecules were immobilized on Poly-L-lysine coated glass slides and visualized using FISH. Subclones as small as 2 kb can be mapped to a specific position in reference to the BAC vector. Several BAC clones derived from rice and sorghum centromeric regions were analyzed by this technique. These centromeric BACs contain complex DNA, including both middle and highly repetitive DNA sequences. The sequence data from these BACs were impossible to be assembled using available software. Using the current technique, we were able to reveal the distribution and organization of different repeats within the centromeric BAC inserts. Our results demonstrate that this technique is particularly valuable to characterize BAC clones containing complex repetitive DNA sequences that are difficult to characterize using restriction mapping or sequencing approaches.