PCR amplification and simultaneous digoxigenin incorporation of long DNA probes for fluorescence in situ hybridization.
Nonradioactive in situ hybridization has found widespread applications in cytogenetics. Basic requirements are DNA probes in sufficient amounts and of high specificity as well as a labeling protocol of good reproducibility. The PCR has been of fundamental importance for the amplification of DNA sequences and thus for the production of DNA probes. Meanwhile, PCR protocols for amplification of DNA have reached a high degree of automation. So far, incorporation of labeled nucleotides into these DNA probes has normally been done by nick translation. Here we show that in using the PCR, amplification of a DNA probe larger than one kilobase accompanied by simultaneous incorporation of digoxigenin-11-dUTP can be performed for in situ hybridization experiments. As an example, the DNA probe pUC 1.77 specific for the subcentromeric region q12 of chromosome number 1 was used and hybridized against metaphase chromosomes from human lymphocytes. The labeled chromosome region was detected by anti-digoxigenin-fluorescein, Fab fragments. The experiments were evaluated by digital image analysis of microphotographs.
Celeda
D
D
Institute of Applied Physics, University of Heidelberg, FRG.
Bettag
U
U
Cremer
C
C
D002878:Chromosomes, Human, Pair 1; D015342:DNA Probes; D003856:Deoxyuracil Nucleotides; D004076:Digoxigenin / Q000031:analogs & derivatives*; D016650:Fluorescein-5-isothiocyanate; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes; D006801:Humans; D007202:Indicators and Reagents; D008214:Lymphocytes / Q000648:ultrastructure; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction
D015342:DNA Probes; D003856:Deoxyuracil Nucleotides; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; C070202:digoxigenin-11-deoxyuridine triphosphate; D016650:Fluorescein-5-isothiocyanate; D004076:Digoxigenin