Introduction to the Workshop
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The electronic form of this document may be cited in the following style:
Human Genome Program, U.S. Department of Energy, DOE Human Genome Program Contractor-Grantee Workshop IV, 1994.
Abstracts scanned from text submitted for November 1994 DOE Human Genome Program Contractor-Grantee Workshop. Inaccuracies have not been corrected.
Bacterial Artificial Chromosomes (BACs) for Mapping and Sequencing
Melvin I. Simon[1,4], Ung-Jin Kim, Hiroaki Shizuya, Bruce Birren, and Jerry Solomon
Biology Division, California Institute of Technology, Pasadena, California. Whitehead Institute, Center for Genome Research, Massachusetts Institute of Technology, Cambridge Massachusetts. Beckman Institute, California Institute of Technology, Pasadena, California. Corresponding author.
Bacterial artificial chromosomes and Fosmids are stable, non-chimeric, highly representative cloning systems. The BACs maintain large fragment genomic inserts (100-300 kilobase pairs) that are easily prepared as DNA templates for sequencing. We have improved the methods for generating BACs and developed extensive BAC libraries. A BAC library in the mouse was prepared in less than two months; it consists of approximately 105,000 clones providing approximately four-fold coverage of the mouse genome. BAC libraries corresponding the human genome now provide more than 6X coverage. Libraries have been made with DNA from many other organisms.
In order to demonstrate the utility of the BAC system for mapping, we have applied and extended a variety of existing techniques to generate a physical map of chromosome 22. We have chromosome-specific-BACs that correspond to approximately three-fold coverage of the chromosome. These BACs have been arranged into contigs and we are currently engaged in closing the remaining gaps to finish a complete map of the 45 megabase Q-arm of chromosome 22 which will incorporate over 100 STS markers, cosmid and cDNA markers and will be correlated with yeast artificial chromosome maps that have been developed in other laboratories for chromosome 22. BACs provide a rapid, easy source for hybridization or PCR-STS mapping that can allow the complete coverage of all of the chromosomes. Furthermore, the BAC libraries represent general resources that can be interrogated with almost any form of DNA probe to generate 1-2 megabase contigs in any region of the genome. This will be illustrated by a number of examples. Furthermore, since the BACs are present as closed covalent circular DNA which is easily isolated from E. coli, the BAC contigs provide convenient stable, ordered sets of material for long range sequencing. The circular form of the plasmid also allows BACs to be easily manipulated by the techniques of bacterial genetics and a variety of transposons can be used to retrofit the BAC vectors with properties that allow specific inserts to be expressed in eukaryotic cells or to generate nested sets of deleted fragments of any genomic insert that provide for the rapid characterization of the insert.
We are currently designing a variety of transposon based methods for sequencing using BACs and Fosmids as well as long range PCR to map transposon inserts. These approaches can be taken together to form an integrated long range sequencing system that can eventually be automated. Progress with these approaches will be described.