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.
YACs with Unique Capture Handles
Lucy L. Ling, Jonathan Norcross, Elizabeth Borsody and Donald T. Moir
Department of Human Genetics & Molecular Biology, Collaborative Research, Inc., Waltham, MA 02154
Yeast artificial chromosomes have been used extensively for physical mapping. However to obtain sequence ready clones, current approaches are to anchor cosmids or P1's representing an entire YAC. Because STS densities even as high as one per 100 kb may be inadequate, this approach will demand considerable effort which could be avoided if YACs could be sequenced directly or subcloned for sequencing. Current YACs are not suitable sequencing templates, mainly due to (1) limited DNA yield because YACs are typically single copy; (2) YAC DNA cannot be separated easily from yeast DNA; and (3) YACs are frequently chimeric. We have focussed on solving these problems.
Recombination within the yeast host strain has been postulated to contribute to the mechanism of chimerism. The contribution of host genetic background to chimera frequency was determined by introducing the same ligation mixture (greater than 400kb) into three isogenic yeast hosts that differ only in genotype at the RAD52 and RAD1 loci. The frequency of chimeras was measured by fluorescent in situ hybridization (FISH) to human prometaphase spreads. Fewer chimeric YACs were found in recombination-deficient hosts, in all size ranges (10% in recombination-deficient hosts compared to 47% in wild-type hosts).
Building on the amplifiable YAC copy number system of Smith et al. (1990, PNAS 87:8242) a new YAC vector, pCGS1000, has been constructed with an EcoR 1 cloning site flanked by unique restriction sites and polypurine tag sites for capture by triple helix interaction. Resulting YACs can be digested with SceI to remove the vector arm releasing only the insert. The copy number of these YACs can be amplified by growth in the presence of methotrexate, sulfanilamide and thymidine. DNA fragments up to 20kb containing these polypurine sequences were captured specifically by triplex affinity to polypyrimidine third strands attached to a solid support. The effect of the location of the polypurine tag on the quality and quantity of capture was analyzed. Vector-YAC PCR products representing both ends of the YAC can be captured selectively by using either the left or right tag polypyrimidine sequences and sequenced directly. We are currently investigating methods for capturing the entire YAC insert.
Supported by DOE Grant DE-FG02-92ER61399