SIGNAL AMPLIFICATION IN THE DETECTION OF SINGLE-COPY DNA AND RNA BY ENZYME-CATALYZED DEPOSITION (CARD) OF THE NOVEL FLUORESCENT REPORTER SUBSTRATE CY3.29-TYRAMIDE

We demonstrate that the CAtalyzed Reporter Deposition method (CARD), u tilizing the novel fluorescent reporter Cy3.29-tyramide, is successful in the Fluorescent In Situ Hybridization (FISH) detection of RNA and single-copy DNA. Histone 4 expression is detected in RNA extracts of S -phase, synchronized HeLa cells by dot-blot analysis. Gene expression of histone 4 in HeLa cells is demonstrated by FISH via CARD, utilizing oligonucleotide probes. Fluorescence intensity measurements on CARD-a mplified histone 4 RNA detection showed (a) a 25-fold amplification of the signal brightness by biotinylated oligonucleotide probes and (b) a sixfold amplification of the signal brightness by horseradish peroxi dase (HRP)-labeled histone 4 probes vs the directly stained control. T he sensitivity of the CARD method is demonstrated by the FISH detectio n of single-copy DNA on human corneal fibroblast and HeLa 53 interphas e nuclei. Chromosomal localization of the single copy DNA is demonstra ted on HeLa 53 metaphase chromosome spreads.