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or from the Human Genome Database (http://gdbwww.gdb.org/)
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Data on microsatellite markers, and the corresponding primer sequences, were accessed through the Cooperative Human Linkage Center (http://www. chlc.org/HomePage.html) or from the Human Genome Database (http://gdbwww.gdb.org/).
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15
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4243065925
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note
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The methods for establishing xenografts and for PCR and multiplex PCR assays were as in (7), and for Southern blots as in (6). STS markers used to exclude the involvement of DCC were SSAV, D18S523, D18S526, D18S101, and the microsatellite marker DCC (15). All PCR reactions were repeated at least three times and confirmed by a second primer pair designed on nearby sequences to exclude the possibility of a primer site polymorphism. The quality of the DNA was further ensured by the successful amplification of a 1.8-kb fragment (exons 5 to 9 of p53) and of numerous primer sets for microsatellite markers.
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17
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84901957018
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in press
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YAC ends were isolated by the inverse PCR technique (28). The amplified ligation fragments were sequenced by cycle sequencing (SequiTerm, Epicentre Technologies, Madison), and 20-mer oligonucleotide pairs for STS markers were designed PCR analysis of monochromosomal somatic ceil hybrid DNA (NIGMS mapping panel 2, Conell Cell Repositories) confirmed STS localization to chromosome 18. The full genomic map is published [S. A. Hahn et al , Cancer Res., in press]
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Hahn, S.A.1
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4243111442
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note
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A PCR-based P1 screening was performed by Genome Systems, St. Louis, with STS markers flanking the consensus deletion. Positive P1 clones from the DuPont Merck Pharmaceutical Company Human Foreskin Fibroblast Library 1 (DMPC-HFF#1) were: 1210-C10, 0960-F5, and 0630-H5. We performed a second screen with human PAC library (purchased from Genome Systems) by hybridizing a random primer-labeled PCR product to gridded PAC library filters
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21
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0028949381
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Partial Nde II-digested YAC DNA was subcloned into the SuperCos-1 vector (Stratagene). Cosmids were screened and identified by PCR with STS markers derived from the region of interest and were sequenced using primers specific for vector sequences. P1-PAC end sequences were generated by direct sequencing or by a PCR-based amplification technique [Y.-G. Liu, R. F. Whittier, Genomics 25, 674 (1995) and L. T da Costa, unpublished data]
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Liu, Y.-G.1
Whittier, R.F.2
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22
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0028949381
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unpublished data
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Partial Nde II-digested YAC DNA was subcloned into the SuperCos-1 vector (Stratagene). Cosmids were screened and identified by PCR with STS markers derived from the region of interest and were sequenced using primers specific for vector sequences. P1-PAC end sequences were generated by direct sequencing or by a PCR-based amplification technique [Y.-G. Liu, R. F. Whittier, Genomics 25, 674 (1995) and L. T da Costa, unpublished data]
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Da Costa, L.T.1
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23
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4243102524
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note
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For exon amplification, DNA from cosmid C917-46 was digested with Bam HI and Bgl II and ligated into the pSPL3 exon-trapping vector (Gibco/BRL). Exontrapped sequences were analyzed by BLAST homology searches The location of the exon-trapped sequences to the region was confirmed by Southern blot analysis of Eco RI-digested DNA of cosmid C917-46. 5′-RACE was performed according to the manufacturer's instructions (Clontech, Palo Alto). cDNA library screening was performed with exontrapped sequences or Eco RI restriction fragments from c917-46 as probes. The cDNA libraries were derived from HeLa cells, human placenta, and human fetal brain (Stratagene), and the human cotorectal cancer cell line SW480 (Clontech).
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24
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4243116038
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note
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These 61 xenografts included 28 from the initial panel and 33 new ones.
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25
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0027763498
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The protein assay was performed with the TNT kit (Promega). Primer sequences (5′ to 3′) were: DPC4S, GGATCCTAATACGACTCACTATAGGGC-CGCCACCATGGCCTGTCTGAGCATTGTGCATAG; and DPC4AS, CAGTTTCTGTCTGCTAGGAG [M W Powell et al., N. Engl. J. Med. 329, 1982 (1993)].
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Powell, M.W.1
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26
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4243152592
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note
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PCR amplification of the exons was performed as in (29) Mutations were confirmed in a second PCR reaction. The PCR primer sequences are available upon request, Intronic primers were generally used to amplify exons from the xenografts grown in mice, because exonic primers generated PCR products from mouse DNA that were identical in size to the human PCR products.
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27
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4243148109
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note
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Direct sequencing of microdissected tumor cells was achieved in two of the six tumors with DPC4 mutations. Contamination of normal cells in primary pancreatic carcinomas usually precludes direct genetic study (7).
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28
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0028940853
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Markowitz, S.1
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4243072632
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note
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We thank K. W. Kinzter and B. Vogelstein for cDNA library clones. Supported by the SPORE in Gastrointestinal Cancer, NIH grant CA62924, Deutsche Krebshilfe (S A H.), and Junta Nacional de Investigaçäo Científica e Technológica Scholarship BD1508/ 91 (L T C ) S.K is a McDonnell Foundation Scholar.
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