YEAST ARTIFICIAL CHROMOSOME-BASED GENOME MAPPING

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Yeast artificial chromosomes (YACs) as a cloning vector provide the basis for an approach to the systematic analysis of the contents of the human genome. YACs can clone much or all of the human genome faithfully in fragments of average size 250 kb or more, and YACs cognate for specific probes can be recovered from libraries and assembled in contigs of average size at least 2 Mb. In work thus far, a collection of YACs containing five genomic equivalents of human DNA has been organized in matrix arrays and screened for cognate YACs in a Core Screening Lab. Screening involves the use of specific primer pairs and the Polymerase Chain Reaction (PCR) on YAC DNAs from pools of colonies, and then detection of individual clones by hybridization of the PCR product as a probe against DNA from the colonies in positive pools. Through the efforts of the Development laboratory in conjunction with Core Units and collaborating laboratories, the P50 Center seeks to increase the efficiency of screening for YACs 3 to 10-fold, and thus to approach three-year goals of 1) screening for up to 1500 YACs covering individual genes and regions of up to 4 Mb as well as 600 YACs specific for chromosome 4 (Project 1); and 2) mapping up to 3000 YACs across each of chromosomes 7 and X (Projects 2 and 3). This throughput could be achieved by screening completely by PCR methods, and by generating primer pairs independent of the YACs themselves. [Primer pairs are to be derived from other venues for other loci; for chromosomes 7 and X, they are to be generated in the STS Core Lab from specialized libraries of 200 to 1000 base pair fragments cloned from flow-sorted chromosomes.] Each primer pair defines a sequence-tagged site [STS, as in Olson et al. (Science 245;1434-1435)] in a cognate YAC. The alignment of YACs, and the organization of the resultant map, is based first on their content of different STS; second, on their relation to the genetic linkage map, as refined in the Linkage Laboratory; and third, on auxiliary physical mapping studies, including the use of jumping clones and in situ hybridization. With the aid of the Information Handling and Analysis Core, the YAC contigs would provide a basis for the merger of genetic and physical markers into a single unified map. The Genome Project relatedness of the Center would thus include provision of about 15% of the unified map, in a format easy to refine, using more efficient and less costly methods than are currently available.