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5 plaques were screened in duplicate using 0.4-kb Eco RI and 0.8-kb Xho I fragments of M2. Of the 12 isolated clones, 6 were subcloned into pBSII (Stratagene). To obtain the full coding sequence, we completely sequenced H2 and partially sequenced H14, H20, and H21. MacVector and GCG programs were used for sequence analysis.
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To perform radiation hybrid mapping of the human PTC gene, we first developed oligonucleotide primers and conditions for specifically amplifying a portion of the gene from genomic DNA by PCR. These primers were designated sequence-tagged sits (STS) SHGC-8725, and PCR generated a 196-bp product that was observed on agarose gel electrophoresis when human DNA was used as template but not when rodent DNA was used as template. We then scored in duplicate for the presence or absence of the 196-bp product in 83 radiation hybrid DNA samples from the Stanford G3 Radiation Hybrid Panel purchased from Research Genetics, Inc. By comparing the pattern of G3 panel scores with those from a series of Genethon meiotic linkage markers, we determined that no radiation hybrid breaks were observed between PTC and D9S287 in the 83 hybrid cell lines. These results indicate that the PTC gene lies within 50 to 100 kb of the marker D9S287. Subsequent physical mapping in YAC and BAC clones confirmed that this close linkage estimate was accurate. Detailed map information can be obtained from http: //www-shgc.stanford.edu
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BAC 192J22 was digested with restriction enzymes and ligated with vectorette linkers [J. Riley et al., Nucleic Acid Res. 18, 2887 (1990)]. Twenty exon-intron boundaries within the coding sequence were determined by sequencing of PCR products amplified using the universal vectorette primer and PTC cDNA primers.
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Mutations in exons 3 and 15 were identified by SSCP analysis of PCR products amplified from genomic DNA. Primers used to amplify exon 3: 5′-GAGTTTGCAGTGATGTTGCTATTC-3′ and 5′-ACCGCCTTACCTGCTGCTC-3′, exon 15: 5′-GGAAGAGTCAGTGGTGCTCC-3′ and 5′-CGCCAAAGACCGAAAGGAC-3′. The PCR products were sequenced on both strands directly from the PCR-produced templates and after cloning into Bluescript.
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We thank the patients and family members who contributed samples and the physicians who helped us obtain patient materials. We also thank J. Fletcher for help in the genesis of the project; R. Gorlin and J. B. Howell for encouragement; J. Hooper for insights and ideas; G. Barsh, S. Kim, D. Kingsley, and A. Oro for comments on the manuscript; X. Zhang for assistance; and M. Nguyen and A. Smith for sequencing. Supported by the Cancer Research Fund of the Damon Runyon-Walter Winchell Foundation Fellowship (DRG 1218) and a Walter Berry Fellowship to R.L.J., NIH grant AR3995 to E.H.E., a Dermatology Foundation Fellowship to J.X., a Medical Research Council Traveling Fellowship to A.G.Q., and a Howard Hughes Medical Institute (HHMI) Predoctoral Fellowship to L.V.G. M.P.S. is an Investigator of the HHMI.
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