STC Production on Human BACs
Several types of DNA library resources were sponsored by the DOE before and during the Human Genome Program (HGP). These included both prokaryotic and eukaryotic vector systems, and clone libraries representing single chromosomes. Bacterial Artificial Chromosomes (BACs) became the most broadly used resource for several reasons. The large size was a good match for capabilities of high throughput sequencing centers. As contrasted to some earlier resources, chimerism (having gene segments from multiple chromosome sites combined in one clone) is substantially if not completely absent. With some interesting exceptions, the BACS are stable in their bacterial hosts. In support of the functional analysis of genes, the BACs are very useful for making transgenic animals with segments of human DNAs. A brief history of BAC development is available in a preface to a 2003 issue of Methods in Molecular Biology, wherein details of BAC related protocols reside.
One particular BAC project was crucial to the timely completion of human genome sequencing. (See history.) In a 1996 initiative, the DOE Office of Biological and Environmental Research sponsored the production of sequence tag connectors (STCs) for the BACs being used in human genome sequencing. (STCs are sequence reads at the ends of cloned DNA segments; they mark the boundaries of the cloned DNA.) This publicly available resource has served both the international public collaboration and Celera Genomics Inc. in the generation of the human genome sequence.
The BACs representing a genome can together serve as a scaffold on which much shorter DNA sequence assemblies can be located. The STCs have particular roles in identifying BACs:
The data resources generated under the STC initiative thus speeded human genome sequencing worldwide. STC generation is now an integral component of most genome sequencing projects.
The target of one STC for every three kilobases of the human genome was achieved during the year 2000. The primary STC production sites were The Institute for Genomic Research (TIGR) and the former Sequencing Center of the Department of Molecular Biotechnology, University of Washington (UWMB), with capabilities now at the Institute for Systems Biology with director Leroy Hood . Much more detail is in this history, or can be provided by Marvin.Stodolsky@science.doe.gov.
Complementary mapping resources
The STC data resource is complemented by several other types of mapping information. FISH (fluoresence in situ hybridization) mapping by J. Korenberg provided the first quality validation of BAC resources. The NCI extramural division, in conjunction with DOE, has awarded a consortium of three grantees to generate a Mapped Human Bacterial Artificial Chromosome (BAC) Clone Resource. Within the consortium, Barbara Trask has extending FISH analyses to newer BAC libraries. FISH mapped BACs with STCs have been added to Radiation Hybrid (RH) maps at Stanford U. RH STS markers and gene-based EST markers served to identify corresponding BACs by teams under Pieter de Jong and Ung-Jin Kim (while formerly at CalTech). Restriction fingerprints of BACs generated at UWMB and with NIH/NHGRI support at the Washington University Genome Sequencing Center aided contig construction and validation, in preparations for sequencing.