Plant, Animal & Microbe Genomes X Conference

January 12-16, 2002
Town & Country Convention Center
San Diego, CA

Workshop: Aquaculture
            

DEVELOPMENT OF A HIGH-THROUGHPUT SYSTEM FOR FINGERPRINTING BAC LIBRARIES OF TILAPIA

¹ Hubbard Center for Genome Studies, University of New Hampshire, 35 Colovos Road ,Durham, NH 03824 USA

To speed the positional cloning of QTL for commercially important traits in tilapia we are planning to collect restriction fingerprints of 30,000 BAC clones, equivalent to 5x coverage of the genome. From these fingerprints we hope to assemble contigs averaging 1 Mb covering 95% of the tilapia genome. We are using two BAC libraries whose average insert sizes are 145 kb and 194 kb. We manually isolate DNA from clones grown in 96 deep-well plates using the method of alkaline lysis. BAC DNAs are cut with combination of two restriction enzymes. A six-base cutter (HindIII or BamHI) creates fragments averaging 4096bp. These fragments are then reduced to an average of 250 bp with a 4-base blunt cutting enzyme (HaeIII or RsaI). Simultaneous with the restriction, fluorescently labelled ddATP (HindIII) or ddGTP (BamHI) are incorporated into the 3' sticky end. These fluorescently-labelled DNA fragments are then separated by capillary acrylamide electrophoresis in a Beckman CEQ2000 DNA sequencer. We collect fragments sizes in the range of 70 to 620 bp, as this produce the optimum balance between number of bands and the speed and accuracy of detection. After a semi-automated analysis, fragment sizes from each clone are imported into the FPC program for identification of overlapping BACs and contig construction. As the project proceeds, fingerprints and contig data will be made available through a WWW interface.