DOE Human Genome Program Contractor-Grantee Workshop VIII
February 27-March 2, 2000  Santa Fe, NM

17. Human and Mouse BAC Libraries for Genome Sequencing, Mapping, and Functional Analysis

Kazutoyo Osoegawa, Chung Li Shu, Aaron Mammoser, Joe Catanese, and Pieter J. De Jong

Department of Cancer Genetics, Roswell Park Cancer Institute, Buffalo, NY 14263 and Children's Hospital Oakland Research Institute, Oakland, CA 94609

pieter@dejong.med.buffalo.edu

Our earlier 25-fold redundant human BAC library (RPCI-11; EcoRI fragments) has recently been expanded with an additional 7-fold genome redundancy from the same donor using MboI-digested DNA. Insert sizes averaged 173 and 195 kb for the early and late parts of the library, respectively. A 1.5-Mb BAC contig was extensively analyzed to test the human BAC library for clonal integrity and fidelity. The results indicate the absence of chimeric clones and 19 rearranged clones in the contig of 169 BACs. Three murine libraries (each 11-fold genome redundant) have previously been constructed various digest strategies, two strains (129S6/SvEvTac and C57BL/6J) and either PAC or BAC vectors. The BAC library for the C57BL/6J strain (designated RPCI-23) is most significant in view of the large EcoRI-inserts (average 200 kb) and because it was selected as a preferential source for murine genome sequencing. To obtain additional representation for the C57BL/6J strain, we created an additional the C57BL/6J BAC library (RPCI-24) from male DNA partially digested with MboI (average insert sizes about 155 kb). To permit the cloning of sheared DNA, a new vector, pTARBAC6, was constructed with two BstXI sites for cloning. The BstXI sites have non-complementary ends to avoid vector self-ligation. Blunt-ended DNA fragment are ligated to a BstXI linker to create ends complementary with the vector. In pilot experiments, fragments were cloned from HincII partially-digested DNA and DNaseI partially-digested DNA resulting in average insert sizes around 100 and 60 kb, respectively. To maximize randomness of the BAC cloning process, we are optimizing the cloning of blunt-ended fragments obtained by shearing. Information on current libraries can be found at http://bacpac.med.buffalo.edu .

Supported by grants from the U.S. DOE (#DE-FGO3-94ER61883) and NIH (#1RO1RGOl 165).