Loss of heterozygosity in normal tissue adjacent to breast carcinomas

Science

Volumn 274, Issue 5295, 1996, Pages 2057-2059

  Deng, Guoren ^a   Lu, You ^a   Zlotnikov, Galina ^a   Thor, Ann D ^b   Smith, Helene S ^a,c  

^a CALIFORNIA PACIFIC MEDICAL CENTER   (United States)

^b NORTHWESTERN UNIVERSITY   (United States)

^c UNIVERSITY OF CALIFORNIA   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

ARTICLE; BREAST CARCINOGENESIS; BREAST CARCINOMA; CANCER STAGING; CHROMOSOME 3P; HETEROZYGOSITY LOSS; HUMAN; PRIORITY JOURNAL;

ALLELES; BREAST; BREAST NEOPLASMS; CARCINOMA, DUCTAL, BREAST; CHROMOSOMES, HUMAN, PAIR 11; CHROMOSOMES, HUMAN, PAIR 13; CHROMOSOMES, HUMAN, PAIR 17; CHROMOSOMES, HUMAN, PAIR 3; DNA; DNA, NEOPLASM; FEMALE; GENE DELETION; HETEROZYGOTE; HUMANS; POLYMERASE CHAIN REACTION; RECEPTOR, ERBB-2; TUMOR SUPPRESSOR PROTEIN P53;

EID: 0030461224     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.274.5295.2057     Document Type: Article

References (30)

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17. 2, 0.01% gelatin, 200 μM each of deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP) and deoxythymidine triphospate (dTTP), 0.1 μM upstream and downstream primers, 1% dimethyl sulfoxide (DMSO), 0.44 μg Thermus aquaticus (Taq) antibody (TaqStart antibody, Clontech), and 2 units of Taq DNA polymerase C [D. K. Wright and M. M. Manos, in PCR Protocols: A Guide to Methods and Applications, M. A. Innes, D. H. Gelfand, J. J. Sninsky, T. J. White, Eds. (Academic Press, San Diego, CA, 1990), pp. 153-158]. The PCR reaction consisted of 45 cycles of denaturing (94°C for 30 s), annealing (56 to 66°C for 30 sec.), and chain extension (72°C for 30 s). The PCR products were separated by electrophoresis [G. Deng et al, Cancer Res. 54, 499 (1994)]. Each allele of the PCR product was measured by densitometry of x-ray film exposed to the gel of of a negative photographic film of the ethidium bromide-stained gel. The density ratios of the two alleles (the undigested allele: the digested allele, or the top allele: the bottom allele) for different tissues were calculated. The density of each allele was determined (using a Computing Densitometer by Molecular Dynamics, Model 300A) and the relative densities of the two alleles in each lane were compared to their relative densities in skin. Samples were scored as positive for LOH if the calculated value was <0.70 according to the following equation: [[density of weaker allele of sample : density of stronger allele of sample/density of weaker allele of skin: density of stronger allele of skin]]. This calculated ratio for each lane in Rg. 2 relative to skin (lane 1) is as follows: Fig. 2A, lane 2, 0.95; lanes 3 to 8, <0.10; Fig. 2B, lane 2, 0.31; lane 3, 0.29; lane 4, 0.37; Rg. 2C, lane 2, 0.15; lane 4, 0.25; lane 6, 0.93.
18. 2, 0.01% gelatin, 200 μM each of deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP) and deoxythymidine triphospate (dTTP), 0.1 μM upstream and downstream primers, 1% dimethyl sulfoxide (DMSO), 0.44 μg Thermus aquaticus (Taq) antibody (TaqStart antibody, Clontech), and 2 units of Taq DNA polymerase C [D. K. Wright and M. M. Manos, in PCR Protocols: A Guide to Methods and Applications, M. A. Innes, D. H. Gelfand, J. J. Sninsky, T. J. White, Eds. (Academic Press, San Diego, CA, 1990), pp. 153-158]. The PCR reaction consisted of 45 cycles of denaturing (94°C for 30 s), annealing (56 to 66°C for 30 sec.), and chain extension (72°C for 30 s). The PCR products were separated by electrophoresis [G. Deng et al, Cancer Res. 54, 499 (1994)]. Each allele of the PCR product was measured by densitometry of x-ray film exposed to the gel of of a negative photographic film of the ethidium bromide-stained gel. The density ratios of the two alleles (the undigested allele: the digested allele, or the top allele: the bottom allele) for different tissues were calculated. The density of each allele was determined (using a Computing Densitometer by Molecular Dynamics, Model 300A) and the relative densities of the two alleles in each lane were compared to their relative densities in skin. Samples were scored as positive for LOH if the calculated value was <0.70 according to the following equation: [[density of weaker allele of sample : density of stronger allele of sample/density of weaker allele of skin: density of stronger allele of skin]]. This calculated ratio for each lane in Rg. 2 relative to skin (lane 1) is as follows: Fig. 2A, lane 2, 0.95; lanes 3 to 8, <0.10; Fig. 2B, lane 2, 0.31; lane 3, 0.29; lane 4, 0.37; Rg. 2C, lane 2, 0.15; lane 4, 0.25; lane 6, 0.93.
19. 2, 0.01% gelatin, 200 μM each of deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP) and deoxythymidine triphospate (dTTP), 0.1 μM upstream and downstream primers, 1% dimethyl sulfoxide (DMSO), 0.44 μg Thermus aquaticus (Taq) antibody (TaqStart antibody, Clontech), and 2 units of Taq DNA polymerase C [D. K. Wright and M. M. Manos, in PCR Protocols: A Guide to Methods and Applications, M. A. Innes, D. H. Gelfand, J. J. Sninsky, T. J. White, Eds. (Academic Press, San Diego, CA, 1990), pp. 153-158]. The PCR reaction consisted of 45 cycles of denaturing (94°C for 30 s), annealing (56 to 66°C for 30 sec.), and chain extension (72°C for 30 s). The PCR products were separated by electrophoresis [G. Deng et al, Cancer Res. 54, 499 (1994)]. Each allele of the PCR product was measured by densitometry of x-ray film exposed to the gel of of a negative photographic film of the ethidium bromide-stained gel. The density ratios of the two alleles (the undigested allele: the digested allele, or the top allele: the bottom allele) for different tissues were calculated. The density of each allele was determined (using a Computing Densitometer by Molecular Dynamics, Model 300A) and the relative densities of the two alleles in each lane were compared to their relative densities in skin. Samples were scored as positive for LOH if the calculated value was <0.70 according to the following equation: [[density of weaker allele of sample : density of stronger allele of sample/density of weaker allele of skin: density of stronger allele of skin]]. This calculated ratio for each lane in Rg. 2 relative to skin (lane 1) is as follows: Fig. 2A, lane 2, 0.95; lanes 3 to 8, <0.10; Fig. 2B, lane 2, 0.31; lane 3, 0.29; lane 4, 0.37; Rg. 2C, lane 2, 0.15; lane 4, 0.25; lane 6, 0.93.
20. The primer pairs included restriction fragment length polymorphisms (RFLPs), C-A repeat, and tetranucleotide microsatellite polymorphic loci. The sequences of the PCR primers for LOH analysis are as follows (in each case, the forward primer is followed by the reverse primer): 3p26 (76) D3S2397: ATAGAGCCACACTTTGTCTCA: TCTTTGAGAAC-CACTGTCTCC; 3p25 (16) D3S1597: AGTACAAATA-CACACAAATGTCTC: GCAAATCGTTCATTGCT; 3p24 (17) EABH: CATCTGAAATGCTGACCTGTT: AGCTGTCAGAACTAAGTGCTT; 3p24 (18) EABMD: AACGTTGGACCTCAAGCCCAT: AGAATGCCAAG-GAAGGGTGCA; 3p24 (19) D3S1244: GTGCCCTTC-CAGGAGTT:AGTGAGGCATCCACTACC; 3p22 (16) D3S1612: TCTTTTAGTCAGCAGTTATGTC: CCC-ATTAAGAAATGTTACTCTAC; 3p22 (76) D3S1582: AGCAGGTACTATGAAAGCCTGT: GGAACAGCCCA-TGGTTCAC; 3p21 (16) D3S1573: TTCATTTTTGCT-TATTAAGATATGC: CCAGTAAATNACAGGGCTAT; 3p21 (16) D3S1295: TGTAGTMTGGTTTCATGGATA-CAC: ATTTTATAAGTTTTGATACCCTCCC; 11p15.5 (20) TH2:CAGCTGCCCTAGTCAGCAC: GCTTCCGA-GTGCAGGTCACA; 13q13 (27) D13S218: GATTT-GAAAATGAGCAGTCC: GTCGGGCACTACGTTATCT; 13q13 (21) D13S219: AAGCAAATATGCAAAATTGC: TCCTTCTGTTTCTGACTTAACA; 17p13.1 (22) TP53.5: CAATGGATGATTTGATGCTG: TGGTAG-GTTTTCTGGGAAGG; 17p13.1 (23) TP53.6: AGGT-CTGGTTTGCAACTGGG: GAGGTCAAATAAGCAG-CAGG; 17p13.1 (24) TP53.8:T: CAGAAGGAAGTAG-GAAGGACTCAG: GAAGAGCCTCGGTTATGGGTAT-ACA.
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30. Supported by National Cancer Institute SPORE grant 2P50CA58207.