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.
Physical Mapping in Preparation for DNA Sequencing
Andreas Gnirke, Regina Lim, Gane Wong, Jun Yu, and Maynard Olson
Department of Molecular Biotechnology, FJ-20, University of Washington, Seattle, Washington 98195
We are concentrating on a set of methods to allow the precise physical mapping of substantial segments of human DNA. Initial mapping objectives are on the order of 1 Mbp, typically available as YAC contigs supported by STS-content maps. Our goal is to develop subclone collections and precise maps from this starting point. These clones and maps are designed to provide direct support for large-scale DNA sequencing.
The core methodology involves a method of restriction-site mapping that we refer to as multiple-complete-digest (MCD) mapping, which we have developed in collaboration with Will Gillett (Washington University Department of Computer Science). MCD mapping starts from fragment-size lists acquired with multiple restriction enzymes for each member of a highly redundant set of randomly generated subclones that collectively span the mapping objective. In general, enzymes with 6-bp specificity are employed when mapping cosmids subcloned from YACs, while enzymes with 4-bp specificity are employed when mapping small-insert clones subcloned from cosmids. Most experience to date involves mapping cosmids subcloned from YACs.
Digests are analyzed on standard agarose gels, which are post-stained with ethidium bromide or thiazole orange and imaged with a Molecular Dynamics FluorImager 575. We have developed software with which to extract fragment sizes from these images automatically. Fragment-size lists are assembled into MCD maps with software developed by W. Gillett. The basic concepts that underlie this software are (1) absolute compatibility between the maps and the fragment-size lists and (2) compatibility between the partial orderings determined independently in each digest domain for the ends of the clone inserts.
The only requirements for applicability of this approach are the ability to create a highly redundant set of subclones of a mapping objective in a vector system that allows complete-digest fingerprinting and the existence of a higher-level physical map of sufficient resolution to allow the contigs produced by MCD mapping to be ordered and oriented. The present emphasis on YAC contigs is simply motivated by the need for a convenient source of deep cosmid coverage of targeted mapping objectives, particularly coverage that is easily generated in order to allow free experimentation with subcloning methods. The long-term goal of this project is to use MCD mapping as the basic engine of an automated system for the analysis of whole chromosomes or genomes. Hence, there is a major emphasis on minimizing the need for expert human intervention in the data collection and analysis.
We have also developed methods based on the RARE (RecA-Assisted- Restriction-Enzyme) cleavage technique to accommodate gaps in these maps that are due to systematic absence of valid subclones from specific subregions. These techniques also allow STS-content maps to be converted to true, distance-calibrated physical maps.