Bacterial Artificial Chromosome (BAC) libraries have provided a core technology of great importance in genomic science. BAC libraries incorporate randomly derived genome fragments of 100 to 250 Kb pairs into a single copy plasmid in E. coli. BAC libraries are especially useful for organisms that are endangered, difficult to obtain or propagate, or pose significant safety issues. ? ? We plan to provide a modular and scalable system to rapidly construct and analyze BAC libraries. Our four aims are to develop: 1) vectors and technology for production of BAC libraries from randomly fragmented DNA; 2) bacterial hosts free from insertion sequences (IS) for library propagation; 3) technologies using phage packaging for library construction; 4) an optical fingerprinting method for physical mapping. ? ? We will validate the new BAC technologies by constructing and analyzing clone libraries from chicken, an important agricultural species and model organism for developmental biology and human disease. The new vectors use direct selection of clones containing inserts and are backward-compatible with previous vectors. The same vector preparation can accept either random-sheared or partially digested DNA. The vectors remain single copy but are conditionally amplifiable, aiding both stability and DNA isolation. Insert DNA is transcriptionally insulated from the vector by terminators, further promoting insert stability. A new IS-free bacterial host will address IS-mediated problems. In previous systems about 1 in 1000 BAC clones contained IS elements originating from the host, interfering with sequence assembly and precluding the use of such clones in transgenic experiments. ? ? Phage-mediated library packaging produces high transfection efficiencies and uniformly sized inserts which greatly facilitate sequence assembly. Phages with headful sizes of 50 kb (lambda), 100 kb (P1) and larger will be evaluated. The phage vectors will retain the features of our BAC vectors including copy amplification. ? ? A new method for fingerprinting BAC clones based on site-specific quantum dot (QD) labeling will be evaluated. QDs have remarkable features, including a 5-10 fold increase in brightness over other fluorescent labels, lack of photobleaching, and a single excitation source for all emission wavelengths. QDs of various colors will be attached to cut-deficient restriction enzymes (cdREs), bound to DNA and visualized by fluorescence microscopy. The order of colors will define a fingerprint that can uniquely identify a region of DNA. Simultaneous visualization of multiple cdREs in their linear order will greatly facilitate assembly of clones into a genome map. A high throughput format using microfabricated arrays will be developed. ? ? This suite of technologies will greatly assist in the construction and analysis of BAC libraries, which in turn will allow increased use of comparative genomics for understanding human disease and pathogens. ? ?
