The goal of Project 1 is to produce high resolution, sequence-ready, cosmid-based maps for about 30 percent of human genome, consisting of chromosomes 11, 20, 21, 10, 8, 13, 14 and 18. Beginning with chromosome 11, the basic approach will start with the low-resolution, YAC-based maps that are presently under construction in several laboratories, including the San Diego Genome Center, and will proceed YAC-by-YAC along the minimum tiling path of the low-resolution maps. The proposed method will involve the following steps to obtain high-resolution, cosmid-based maps: A given target YAC will be separated from host chromosomes on a pulsed-field gel, extracted from the gel, and radiolabeled by random-hexamer priming for use as a hybridization probe to identify YAC-specific cosmids in arrayed chromosome-specific cosmid libraries by grid hybridization. Cosmid DNAs will be prepared by an automated implementation of the alkaline-lysis procedure using a robotic system already developed in the San Diego Genome Center (the Prepper, Ph.D. DNA prep robot developed by Dr. Garner at General Atomics) and cosmid contigs will be assembled on the basis of restriction analysis. Restriction maps of the cosmids will be determined by an indirect-end-label/partial-digestion method based on excision of the insert plus small amounts of flanking vector DNA by means of NotI sites present in the vector, partial digestion with an enzyme such as EcoRI, and gel-transfer hybridization with oligonucleotide probes specific to the T3 and T7 vector sequences present at each end of the excised NotI fragment. The electrophoresis and gel-transfer hybridization steps will be carried out using the Labimap automated gel analysis system developed at Genethon. Assembly of the contigs will be on the basis of overlapping restriction maps. Contigs from 10 to 20X in depth will be constructed and will cover at least 90 percent of each YAC clone. Gaps in these maps, estimated to be less than 10 kb and spaced on average every 500 kb, will be filled using bacteriophage, lambda, P1 or BAC clones isolated form genomic libraries. Alternatively, if gaps re less than 10 kb, they may be filled by direct amplification from human genomic DNA by PCR using primers produced from the contig ends flanking the gap. Before use for high-resolution mapping, existing cosmid and YAC libraries will be extensively characterized by fluorescence in situ hybridization, and detailed analysis of a sampling of clones carried out to assess quality and representation of the library. Where necessary, these libraries will be supplemented with additional chromosome-specific libraries produced by this project to bring quality and depth into the required range. Dr. Evans considers the rate-limiting step in this procedure to be the gel-transfer hybridization. Using 4 of the Genethon laboratory stations and a production staff of 15, a sustained throughput of 40 YACs per month is anticipated. Projected over the 5-year duration of the project, this throughput would extrapolate to the 1 gigabase pair scale required to complete maps for 30 percent of the human genome.
