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RDA was performed as described in N. A. Lisitsyn et al., (ibid., p. 151). Diploid and aneuploid nuclei from primary breast cancer cells were separated with a fluorescence-activated cell sorter. DNA (100 ng) from each fraction was digested with Bgl II and used to prepare amplicons for 12 separate RDA reactions. Probe CY17 was isolated from one of these reactions. CY17 was 236 bp long and was present in the diploid but not in the aneuploid amplicon from which it was derived. Hybridization of CY17 to normal genomic DNA samples digested with Bgl II revealed no evidence of restriction length polymorphism.
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1842318614
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note
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The CEPH (Centre d'Etude du Polymorphisme Humain) B library (Research Genetics, Huntsville, AL) was screened by PCR using a series of tiered pools to identify unique clones.
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-
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14
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1842382244
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note
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32P]ATP and used to amplify 40 ng of genomic DNA. The samples were then subjected to electrophoresis and autoradiography. The samples included 25 human breast tumor cell lines available from American Type Culture Collection (ATCC) as well as 40 human primary breast tumors xenografted into nude mice. The cell lines were HS578T, SK-BR-3, UACC812, UACC893, MDA-MB-453, MDA-MB-175-VII, MDA-MB468, MDA-MB-361, MDA-MB-231, MDA-MB-436 MDA-MB-415, MDA-MB-330, MDA-MB-157, MDA-MB-134-VI, MDA-MB-435S, ZR 75-30, ZR 75-1, BT-549, BT-483, T-47D, BT-474, DU4475, CAMA1, MCF7, and BT-20.
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15
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0029929955
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U. Kim et al., Genomics 34, 213 (1996). Clones were isolated from a BAC library (Research Genetics) using AFMA086WG9 as an STS probe.
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Genomics
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Kim, U.1
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16
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1842304291
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note
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33P]ddNTP cycle sequencing kit (Amersham). STS primers were designed and the relative location of the STSs determined by testing for their presence in the BAG contig. Primer sequences are available upon request.
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17
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1842355332
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note
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The glioblastoma lines included U105, U118MG, A172, DBTRG-05MG, U373MG, T-98G, U-87MG, and U138MG and 34 glioblastoma xenografts. The prostate cancer cell lines tested were DU145, LN-CaP, NCI H660, and PC-3, and microsatellite analysis revealed that each was unique. With the exception of U105, all lines were obtained from ATCC.
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18
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1842400770
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note
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DNA (10 μg) was digested with Eco RI, resolved on a 1% agarose gel, and transferred to nylon. The JL25 3-kb probe and the 2-kb control probe were randomly labeled and hybridized to the blot consecutively.
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19
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0025860287
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A. J. Buckler et al., Proc. Natl. Acad. Sci. U.S.A. 88, 4005 (1991); G. Lennon, C. Auffray, M. Polymeropoulos, M. B. Soares, Genomics 33, 151 (1996). BACs C and D were digested with Bam HI, BgL II, or both enzymes and then ligated into the trapping vector pSPL3. Libraries were transfected into COS1 cells with lipofectamine and polyadenylated RNA was extracted after 2 days. An exon trapping kit was purchased from GIBCO/BRL.
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Buckler, A.J.1
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A. J. Buckler et al., Proc. Natl. Acad. Sci. U.S.A. 88, 4005 (1991); G. Lennon, C. Auffray, M. Polymeropoulos, M. B. Soares, Genomics 33, 151 (1996). BACs C and D were digested with Bam HI, BgL II, or both enzymes and then ligated into the trapping vector pSPL3. Libraries were transfected into COS1 cells with lipofectamine and polyadenylated RNA was extracted after 2 days. An exon trapping kit was purchased from GIBCO/BRL.
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Lennon, G.1
Auffray, C.2
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1842282283
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note
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The map panel #2 monochromosome panel was purchased from the National Institute of General Medical Science (NIGMS) Human Mutant Genetic Cell Repository.
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1842363115
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STSs Not-5′, PTPD, and ET-1 were amplified from primary glioblastoma DNA and blood DNA and the exonic regions were sequenced.
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1842356306
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note
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We thank B. Vogelstein, N. Tonks, and E. Marcantonio for their comments and S. Kalachikov and R. Hauptschein for helpful suggestions. R.P. is a James S. McDonnell Scholar. M.H.W. is an American Cancer Society Research Professor and is supported by the Department of the Army (DAMD 17-94-I4247), NCI (5R35 CA39829), Amplicon Corporation, and the "1 in 9" breast cancer organization. This work is dedicated to the memory of Richard K. Parsons and Richard P. Sanchez.
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