Towards a globally integrated, sequence-ready BAC map of the human genome
Ung-Jin Kim, Hiroaki Shizuya, and Melvin I. Simon
Division of Biology, 147-75, California Institute of Technology, Pasadena, CA 91125
BACs and Fosmids are stable, non-chimeric, highly representative cloning systems. The BACs maintain large fragment genomic inserts (100-300 kb) and their DNA is easily prepared for most types of experiments including DNA sequencing. We have been improving BAC cloning techniques and constructed > l0X human and mouse BAC libraries. As BACs are proving to be the most efficient reagents for genomic sequencing, we intend to increase the depth of the library up to 30X genomic equivalence to be able to construct optimal contig maps from which one could select minimally overlapping BAC sets for genomic sequencing. The possibility of using BACs as a generalized tool to build a global physical map was explored in our on-going chromosome 22 mapping project. Approximately 700 mapped markers including cDNAs, ESTs, STSs, cosmids, Fosmids, and other landmarks were used to screen the first 4X library. The density of the landmarks in this approach was approximately 1 per every 50-60 kb stretch of chromosome 22q. Many of these markers have been ordered on the YAC-based framework map, allowing rapid and precise localization of BAC contigs along the long arm of chromosome 22. Over 80% of the chromosome has been covered by BACs that have been identified and mapped to corresponding loci by markers. We currently have more than 1,000 chromosome 22-specific BACs, or on the average 3X coverage of chromosome 22q, which are now being characterized by restriction fingerprint analysis and the extent of overlaps between the clones in the contigs determined. Closure of gaps is being sought by screening deeper BAC library with markers and BAC end probes.
Currently large numbers of human genes that have been discovered and exist in the form of sequence-tagged cDNAs or ESTs are being assigned to genomic subregions via YACs and radiation hybrids. Because the landmarks from the YAC framework map have allowed rapid assembly of BAC maps on the chromosome 22q arm, it is feasible to employ the ESTs from the radiation hybrid/YAC frameworks as landmarks and rapidly assemble BACs to generate genome-wide BAC contig maps. Approximately 30,000 such landmarks will correspond to a density of 1 landmark in less than 100 kb of euchromatin. We are planning to utilize initially 30,000 mapped ESTs or cDNAs to construct BAC contigs on the entire genome. The resulting BAC-EST maps, even before its completion, will provide high resolution EST (or gene) maps, and more importantly, entry points for gene finding and large scale genetic sequencing.
* Supported by a Department of Energy grant # FG0389ER60891.
 Shizuya, H., Birren, B., Kim, U.-J., Mancino, V., Slepak, T., Tachiiri, Y., and Simon, M.I. (1992)
Proc.Natl. Acad. Sci. USA 89, 8794-8797.
Abstract scanned from text submitted for January 1996 DOE Human Genome Program Contractor-Grantee Workshop.
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