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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.
Human cDNA Mapping by Hybridization to High-density megaYAC Dot Blots.
Lucy Ling, Betty Borsody, JoAnn Dubois, Jonathan Norcross, Kathy Falls, Romina Bashirzadeh, Liam Haveran, Teresa Kanjuparamban, Ron Lundstrom, and Donald T. Moir
Collaborative Research, Inc, Waltham, MA 02154
The emerging YAC-based physical map of the human genome provides a unique substrate for assignment of genes to chromosomal locations. The resulting gene map will be useful for positional cloning strategies and for improving the resolution of comparative mapping between organisms such as mouse and human. We are developing technology for high throughput, large scale mapping of human cDNAs by hybridization to high density filter grids of DNA from megaYAC clones. Our approach involves redundant pooling of megaYAC DNAs and of cDNA probes to reduce errors and save labor. For example, two 96-well source plates of megaYAC DNA are pooled into one 96-well target plate in a manner which represents each clone in three different (r=3) pools of six clones each (p=6). This reduces the number of required dots by a factor of two (p/r) but at the same time provides multiple signals for each authentic hybridization to a megaYAC. Probes are also pooled in a similar manner to reduce the number of hybridizations required. We have completed isolation of about 100 ug of total yeast DNA from each of 9,000 megaYAC clones (CEPH plates 887-980) to provide 3-fold coverage of the human genome. Despite the chimeric nature of many megaYACs, calculations indicate that 62% of cDNAs will be mapped unambiguously to the correct chromosomal location by hybridization to megaYACs representing 3 human genome equivalents. Multiple possible chromosomal locations, including the correct location, will be obtained for most of the remaining probes. Results from probing of filters containing DNA from 3,000 megaYAC clones (one genome equivalent) in a 3x3 array will be described. Random cDNA probes (provided by G. Lennon) were from a normalized human infant brain cDNA library prepared by Bento Soares (Columbia U.), gridded by Greg Lennon (Livermore), and sequenced by Charles Auffray (Genethon). Probes for known members of multi-gene families were used to examine the extent of cross hybridization. Positive dot blot signals are translated into unique megaYAC addresses by computer, and genetic/physical map positions of resulting megaYACs are determined from the Genethon QUICKMAP database and the Whitehead Institute/MIT Center for Genome Research data releases. Experiments were designed to address the following questions: (1) What is the rate and accuracy of cDNA mapping achievable with this approach? (2) How interpretable are results obtained with probes representing members of homologous multi-gene families? For example, can the multitude of hybridization signals obtained from such probes be disambiguated into unique megaYAC addresses? And, are signals resulting from hybridization to the authentic gene distinguishable from signals resulting from hybridization to related gene family members? (3) Is it possible to map cDNA probes containing repetitive elements by pre-reassociation of the probe with Cot-l DNA prior to hybridization? (4) Can low-stringency inter-Alu element PCR products from megaYAC DNA be substituted for total yeast DNA from megaYAC clones as a substrate for cDNA mapping by hybridization? What fraction of cDNA probes find hybridization targets among the inter-Alu PCR products of megaYACs? Answers to these questions will determine the feasibility of this approach for rapid construction of a total human gene map. This work was supported by DOE grant DE-FG02-92ER61399.