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8 Identification and characterization of homogeneously staining regions by in situ nick translation using restriction endonucleases
Abstracts
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METHODS FOR NON-RADIOACTIVE IN SITU HYBRIDIZATION. JCAG Wiegant, AK Raap, M van der Ploeg. Department of Cytochemistry and Cytometry, University of Leiden, The Netherlands
Non-radioactive in situ hybridizations offer the possibility of rapid and high resolution detection of nucleic acid target sequences in individual cells in an users friendly way. The principles of three of those methods, in which DNA- (or RNA-) probes are enzymatically or chemically modified with haptens (or biotin), are presented. They involve the Bisulfite catalyzed transamination method [1], the Acetyl aminofluorene (AAF) method [2, 3] and the Biotin method [4, 5]. They make use of the possibility to visualize the hybrids with specific antibodies or avidin and second antibodies conjugated with fluorescent or enzyme labels. Since there are available three different fluorescent labels (the new blue immunofiuorochrome AMCA, FITC and TRITC) one is able to simultaneously detect the presence or absence of two or three DNA sequences i'n one nucleus. The value of these methods for (cancer) cytogenetics, e.g. single copy gene localization or detection of numerical chromosome aberrations in interphase nuclei, will be illustrated. REFERENCES 1. Sverdlov et al. [1974): Biochim Biophys Acta 340:153-165. 2. Landegent et al. (1984): Exp Cell Res 153:61-72. 3. Landegent et al. (1985): Nature 317:175-177. 4. Leafy et al. (1983): Proc Natl Acad Sci USA 80:4045-4049. 5. Forster et al. (1985): Nucleic Acids Res 13:745-761.
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IDENTIFICATION AND CHARACTERIZATION OF HOMOGENEOUSLY STAINING REGIONS BY IN SITU NICK TRANSLATION USING RESTRICTION ENDONUCLEASES. S. Bartnitzke, *M. E. Herrmann, D. Eberhardt, **K.-I. Hutter, and J. Bullerdiek. Center of Human Genetics and Genetic Counselling, *University Hospital Rudolf-Virchow St/Ch, Surgical Clinic, Free University of Berlin, Spandauer Damm 130,1000 Berlin 19. **German Cancer Research Center, Project Group Cytometry, lm Neuenheimer Feld, 6900 Heidelberg, FRG.
The DNA of human chromosomes prepared on slides according to routine methods can be nick translated using DNase I and DNA polymerase. Applying a modified method, nonradioactive labelling (biotin-dUTP) is less time consuming and offers a better resolution of the labelling patterns when compared to autoradiographic procedures. The sequence specificity of the restriction endonucleases makes the method well suited for identification and characterization of homogeneously staining regions (HSR) in human cancer cells. For our experiments, we used metaphase preparations and flow-sorted chromosomes from a subline of the human colon carcinoma cell line COLO320 containing two large HSR chromosomes. The HSR had a higher sensitivity against cleavage by EcoRI. However, when comparing the labelling patterns obtained by Mspl and Hpall, we found no evidence that the HSR can be distinguished from other chromosomes by hypomethylation. Nevertheless, a modified procedure will be developed allowing the detection of minor differences as well.
IN SITU HYBRIDIZATION OF NON-RADIOACTIVEREPEATED AND UNIQUE SEQUENCES: APPLICATION TO CANCER CYTOGENETICS. E, Viegas-P6quignotI, M. Jeanpierre2, B. DutrillauxI, F. Apiou ~, H. Magdelenat1, M. Coppey-Moisan1,~. 1. Institut Curie, 26 rue d'Ulm, 75231 Paris C6dex 05. 2. lnstitut de Pathologie Mol6culaire, 24 rue Faubourg St-Jacques, 75674 Paris C6dex 14.3. Institut Curie, 91405 Orsay C6dex, France. In situ hybridization with non-radioactive probes is a rapid and sensitive method for assignment of genes or DNA sequences on metaphase chromosomes and for detection of numerical chromosome abnormalities on interphase nuclei. Our recent results show that short single copy genes or sequences can be accurately mapped on human chromosomes by indirect immune fluorescence. Probe localization can be achieved by classical microscopy or by means of amplified fluorescence digital imaging microscopy. In situ hybridization of a new lq specific probes (Jeanpierre et al., HGM9,1987} to interphase nuclei or breast and colon adenocarcinemas indicate that interphase hybridization is a valid complement to karyotype analysis. Moreover, as lq polysomy is the most frequent chromosomal change observed in solid tumors, interphase hybridization may be a useful tool to separate near diploid tumoral nuclei from normal diploid ones by cell sorting methods in view of molecular analysis.
155
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METHODS FOR NON-RADIOACTIVE IN SITU HYBRIDIZATION. JCAG Wiegant, AK Raap, M van der Ploeg. Department of Cytochemistry and Cytometry, University of Leiden, The Netherlands
Non-radioactive in situ hybridizations offer the possibility of rapid and high resolution detection of nucleic acid target sequences in individual cells in an users friendly way. The principles of three of those methods, in which DNA- (or RNA-) probes are enzymatically or chemically modified with haptens (or biotin), are presented. They involve the Bisulfite catalyzed transamination method [1], the Acetyl aminofluorene (AAF) method [2, 3] and the Biotin method [4, 5]. They make use of the possibility to visualize the hybrids with specific antibodies or avidin and second antibodies conjugated with fluorescent or enzyme labels. Since there are available three different fluorescent labels (the new blue immunofiuorochrome AMCA, FITC and TRITC) one is able to simultaneously detect the presence or absence of two or three DNA sequences i'n one nucleus. The value of these methods for (cancer) cytogenetics, e.g. single copy gene localization or detection of numerical chromosome aberrations in interphase nuclei, will be illustrated. REFERENCES 1. Sverdlov et al. [1974): Biochim Biophys Acta 340:153-165. 2. Landegent et al. (1984): Exp Cell Res 153:61-72. 3. Landegent et al. (1985): Nature 317:175-177. 4. Leafy et al. (1983): Proc Natl Acad Sci USA 80:4045-4049. 5. Forster et al. (1985): Nucleic Acids Res 13:745-761.
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IDENTIFICATION AND CHARACTERIZATION OF HOMOGENEOUSLY STAINING REGIONS BY IN SITU NICK TRANSLATION USING RESTRICTION ENDONUCLEASES. S. Bartnitzke, *M. E. Herrmann, D. Eberhardt, **K.-I. Hutter, and J. Bullerdiek. Center of Human Genetics and Genetic Counselling, *University Hospital Rudolf-Virchow St/Ch, Surgical Clinic, Free University of Berlin, Spandauer Damm 130,1000 Berlin 19. **German Cancer Research Center, Project Group Cytometry, lm Neuenheimer Feld, 6900 Heidelberg, FRG.
The DNA of human chromosomes prepared on slides according to routine methods can be nick translated using DNase I and DNA polymerase. Applying a modified method, nonradioactive labelling (biotin-dUTP) is less time consuming and offers a better resolution of the labelling patterns when compared to autoradiographic procedures. The sequence specificity of the restriction endonucleases makes the method well suited for identification and characterization of homogeneously staining regions (HSR) in human cancer cells. For our experiments, we used metaphase preparations and flow-sorted chromosomes from a subline of the human colon carcinoma cell line COLO320 containing two large HSR chromosomes. The HSR had a higher sensitivity against cleavage by EcoRI. However, when comparing the labelling patterns obtained by Mspl and Hpall, we found no evidence that the HSR can be distinguished from other chromosomes by hypomethylation. Nevertheless, a modified procedure will be developed allowing the detection of minor differences as well.
IN SITU HYBRIDIZATION OF NON-RADIOACTIVEREPEATED AND UNIQUE SEQUENCES: APPLICATION TO CANCER CYTOGENETICS. E, Viegas-P6quignotI, M. Jeanpierre2, B. DutrillauxI, F. Apiou ~, H. Magdelenat1, M. Coppey-Moisan1,~. 1. Institut Curie, 26 rue d'Ulm, 75231 Paris C6dex 05. 2. lnstitut de Pathologie Mol6culaire, 24 rue Faubourg St-Jacques, 75674 Paris C6dex 14.3. Institut Curie, 91405 Orsay C6dex, France. In situ hybridization with non-radioactive probes is a rapid and sensitive method for assignment of genes or DNA sequences on metaphase chromosomes and for detection of numerical chromosome abnormalities on interphase nuclei. Our recent results show that short single copy genes or sequences can be accurately mapped on human chromosomes by indirect immune fluorescence. Probe localization can be achieved by classical microscopy or by means of amplified fluorescence digital imaging microscopy. In situ hybridization of a new lq specific probes (Jeanpierre et al., HGM9,1987} to interphase nuclei or breast and colon adenocarcinemas indicate that interphase hybridization is a valid complement to karyotype analysis. Moreover, as lq polysomy is the most frequent chromosomal change observed in solid tumors, interphase hybridization may be a useful tool to separate near diploid tumoral nuclei from normal diploid ones by cell sorting methods in view of molecular analysis.