Plant & Animal Genome VI Conference

P38

LARGE INSERT BAC AND PAC LIBRARIES FOR MANY SPECIES

Human Genetics Department, Roswell Park Cancer Institute

We have used our P1-derived artificial chromosome (pCYPAC2, pPAC4) and Bacterial Artificial Chromosome (pBACe3.6) vectors to create 10-20 fold redundant large-insert libraries for many animal species, including human, mouse, rat, chimpanzee, baboon and dog. Libraries for cat, insects (Drosophila), Nematodes (C. briggsae), parasites (Plasmodium falciparum), single cellular organism (e.g. Dictyostelium), bacteria (e.g. Legionella) and various agricultural species (animal & plant) are currently in the cloning pipeline. Most of our mammalian libraries, except mouse and rat were prepared using DNA extracted from white blood cells. The average size of inserts in our animal libraries varies from 120 kbp in our initial human PAC libraries to 240 kbp in our most recent BAC libraries. Very (A+T)-rich genomes (e.g. Plasmodium falciparum) could only be cloned with great difficulty and largest average clone sizes were around 8-10 kbp. In the process of preparing the BAC and PAC libraries, we have streamlined the cloning procedures and have introduced many improvements to maintain better integrity of high molecular weight DNA. Pre-electrophoresis of immobilized high-molecular DNA prior to restriction removes inhibitors and therefore results in more homogeneous partial restriction digest conditions. A new double-sizing procedure of restriction fragments in a single agarose gel results in less contamination of the libraries with small insert clones. Electro-elution of the sized DNA fragments from agarose slices provides better recovery and integrity of large DNA (Amemiya and coworkers). Concentration of ligation products by drop dialysis allows a 3-fold increase in the number of colonies obtained. As a result of the improved methods, inserts averaging about 180-240 kb have been obtained in the most recent BAC libraries. See also our website at: http://bacpac.med.buffalo.edu. Supported by grants from the U.S. DOE (#DE-FG03-94ER61883), NIH (#1R01RG01165) NIH (1RO1HL55700-02) and the Burroughs Welcome Fund.