Construction of a BAC Contig Map of Chromosome 16q by Two-Dimensional Overgo Hybridization

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Cold Spring Harbor Laboratory Press

RESUMO

We have used sequence-based markers from an integrated YAC STS-content/somatic cell hybrid breakpoint physical map and radiation hybrid maps of human chromosome 16 to construct a new sequence-ready BAC map of the long arm of this chromosome. The integrated physical map was generated previously in our laboratory and contains 1150 STSs, providing a marker on average every 78 kb on the euchromatic arms of chromosome 16. The other two maps used for this effort were the radiation hybrid maps of chromosome 16 from Whitehead Institute and Stanford University. To create large sequenceable targets of this chromosome, we used a systematic approach to screen high-density BAC filters with probes generated from overlapping oligonucleotides (overgos). We first identified all available sequences in the three maps. These include sequences from genes, ESTs, STSs, and cosmid end sequences. We then used BLASTto identify 36-bp unique fragments of DNA for overgo probes. A total of 906 overgos were selected from the long arm of chromosome 16. Hybridizations occurred in three stages: (1) superpool hybridizations against the 12× coverage human BAC library (RPCI-11); (2) two-dimensional hybridizations against rearrayed positive BACs identified in the superpool hybridizations; and (3) pooled tertiary hybridizations for those overgos that had ambiguous positives remaining after the two-dimensional hybridization. For the superpool hybridizations, up to 236 overgos have been pooled in a single hybridization against the 12× BAC library. A total of 5187 positive BACs from chromosome 16q were identified as a result of five superpool hybridizations. These positive clones were rearrayed on membranes and hybridized with 161 two-dimensional subpools of overgos to determine which BAC clones were positive for individual overgos. An additional 46 tertiary hybridizations were required to resolve ambiguous overgo–BAC relationships. Thus, after a total of 212 hybridizations, we have constructed an initial probe-content BAC map of chromosome 16q consisting of 828 overgo markers and 3363 BACs providing >85% coverage of the long arm of this chromosome. The map has been confirmed by the fingerprinting data and BAC end PCR screening.

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