Abstract
A physical map of overlapping clones covering the human genome will provide a substrate for rapid, high-throughput, high resolution mapping of genes. Such a map of megaYAC clones is currently under development in several laboratories. To permit utilization of this resource, we are developing technology for mapping of cDNAs by hybridization to gridded arrays of DNA from megaYAC clones. Included in this approach are methods and instrumentation for reducing false negatives and false positives by pooling of megaYAC DNAs, for reducing the number of hybridizations by pooling of cDNA probes, and for automating the hybridization and detection steps. Results from a pilot project involving megaYACs representing about one-quarter of the human genome are described. Total yeast DNA was prepared from 730 megaYACs, diluted to a uniform concentration, and pooled with a representation of three and a pool size of three. Various amounts of pooled DNAs from megaYAC clones were gridded onto nylon filters in a medium density array. PCR amplified inserts from cDNA libraries were radiochemically labeled by random priming and used as probes. The sensitivity of detection was adequate even at the lowest megaYAC quantity per dot, i.e., about 0.08 μg of total yeast DNA from each YAC in each dot. Most signals were triplets as expected from the pooling strategy, and the signal intensity of dots was quite uniform. Filters could be re-probed at least five times with no detectable degradation of signal to noise ratio. Application of a “two out of three” rule for validity of “real” signals appears adequate since it permitted accurate identification of all control YACs on the filters. These results suggest that such a hybridization-based approach will permit accurate, rapid, high throughput mapping of cDNAs to intervals in the emerging YAC contig map.
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Moir, D.T. et al. (1994). Mapping cDNAs by Hybridization to Gridded Arrays of DNA from YAC Clones. In: Hochgeschwender, U., Gardiner, K. (eds) Identification of Transcribed Sequences. Springer, Boston, MA. https://doi.org/10.1007/978-1-4615-2562-2_25
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DOI: https://doi.org/10.1007/978-1-4615-2562-2_25
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