| Research Abstract |
A sequence-ready map or the human genome will be generated from PAC contigs because they have provided unique materials for high resolution map and sequencing. We have developed rapid screening and chromosome walking with which almost all gaps were able to be sealed with use of a half million PAC clones, representing approx. 20-fold equivalents of the total genome.
We extracted 1440 DNAs from 384-well plates which have been arrayd with PAC clones, and prepared "PCR screening kits" for the plate selection. For the isolation of clone from the plate, we devised "ditch plates" for mixing bacteria for PCR with which enabled us to isolate any PAC with STS or PCR-primers within a couple of days. For chromosome walking, we applied direct sequencing from the ends of PAC inserts and read consistently more than 500 bases from which we designed PCR-primers for the confirmation of the existing contigs and screening of next PACs. With this system, we have completed a 7-Mb high resolution map from SOD
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l to ETS2. The full integrity of PAC and high dense cosmid overlap map shows the fidelity in cloning with the bacterial system.
Approximately 60 % of the expressed genes of the human genome is known to be associated with CpG islands, most of which are able to be detected with BssHII, EagI and SacII.To help isolate and identify genes, we have sought CpG-islands in the cosmid/PAC contigs with these diagnostic enzymes, assuming close clustering (within l kb) of two or more cutting sites as potential CpG islands. The sequences around the sites have revealed known and unknown genes in this region. Of those, the sequences in PAC 25P16 clone identified.CBR and its analogue, respectively. To verify this approach, we completed the sequence of this clone in shotgun strategies and defined seven CpG islands with which three known and three unknown genes were associated. This approach, designated as CpG-tagged sequence, is rapid and convenient for searching genes and will provide a scaffold for the construction of a transcription map. Less
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