Worm Breeder's Gazette 14(4): 14 (October 1, 1996)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
|1||4444 Forest Park Blvd., St. Louis MO 63108|
|2||Wellcome Trust Genome Campus, Hinxton, Cambs CB10 1SA, UK.|
We have constructed C. elegans and C. briggsae genomic DNA libraries in the pFOS1 "fosmid" vector system. Fosmids are low copy number cosmid-like vectors based on the E. coli F factor replicon (Kim et al. 1992 NAR 20 1083 - 1085). In our hands fosmid clones offer two main advantages. First, randomly selected clones appear to undergo deletion events at a rate much reduced compared to similarly chosen cosmid clones. Second, the yields of DNA obtained from fosmids, while reduced compared to some cosmids, appear reproducible between clones. Both libraries were constructed by ligation of Nde II partially digested genomic DNA into BamHI digested pFOS1 followed by packaging into lambda phage and plating on XL1 Blue MR cells. Approximately 18,000 clones from each library were individually picked into 384 well plates for preservation in glycerol, and these clones were then used to construct high density filters for hybridization. Each filter is 22 cm square and contains all 18,000 clones (approximately 7 X coverage for the genome) with each clone spotted twice on the filter to control for false positives and background. Robotic picking of the clones into 384 well format and construction of the high density filters were performed courtesy of Genome Systems Inc. (St. Louis) who are offering both the high density filters and hybridization based screening of them as services. Interested labs can contact Genome Systems at email@example.com. The C. elegans fosmid library has been characterized by fingerprint analysis of clones selected at random and by direct probing of the high density filters using probe DNAs donated by C. elegans researchers or generated in our labs. Fingerprint data has been incorporated into ACeDB. Fosmid clones are designated in the database by the letter "H". As a result of our analysis, fosmid clones have been identified which extend into gaps in both cosmid-dense and cosmid-poor regions. However, our results indicate that some regions not represented in cosmid clones will not be represented in fosmid clones. Additionally, stable fosmids replacing deleted cosmids have been recovered. As we continue to characterize the library and identify clones for sequencing we are willing to receive probe DNAs from the community and to fingerprint fosmid clones identified in other labs. During characterization of the C. briggsae fosmid library we identified a number of C. briggsae fosmid clones using C. elegans probe DNAs provided by, among others, P. Hoppe, C. Hresko and P. Tan. Probe DNAs were used in hybridization experiments to the high density filters described above and resulting positive clones were fingerprinted and clones selected for sequencing. These sequences will, as with C. elegans sequences, be available on our WWW site (http:// genome.wustl.edu/gsc/). As part of our larger effort to construct a physical map of the C. briggsae genome and generate several megabases of C. briggsae genomic sequence we are willing to consider requests from the community for assistance in mapping and sequencing cosmid or fosmid clones. As a cautionary note, a small number (~15) of the gridded clones appear to be contaminated with vector DNA; we have observed that the cleanest hybridization results to the high density filters are achieved with probe DNAs that are completely free of vector. Questions concerning the C. briggsae fosmid library should be directed to Marco Marra (firstname.lastname@example.org). Questions concerning the C. elegans fosmid library should be directed to Stephanie Chissoe (email@example.com). For a fosmid DNA isolation protocol see http://genome.wustl.edu/gsc/manual/protocols/ - BAC / PAC / Fosmid prep.