Vertebrate chromosomes terminate in variable numbers of T2AG3 nucleotide repeats. In order to study telomere repeats at individual chromosomes, we developed novel, quantitative fluorescence in situ hybridization procedures using labeled (C3TA2)3 peptide nucleic acid and digital imaging microscopy. Telomere fluorescence intensity values from metaphase chromosomes of cultured human hematopoietic cells decreased with the replication history of the cells, varied up to six-fold within a metaphase, and were similar between sister chromatid telomeres. Surprisingly, telomere fluorescence intensity values within normal adult bone marrow metaphases did not show a normal distribution, suggesting that a minimum number of repeats at each telomere is required and/or maintained during normal hematopoiesis.

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We have used horseradish peroxidase-labeled 40 mer oligodeoxynucleotides (HRP-ODNs) specific for the human cytomegalovirus immediate early gene (HCMV-IE) and a novel dinitrophenol-tyramide signal amplification reagent (DNP-TSA plus) to evaluate their utility in fluorescence in situ hybridization (FISH). For DNA FISH, single or cocktails of HRP-ODNs were hybridized to metaphase chromosomes of rat 9G cells which, as determined by DNA fiber FISH, carry an integrated tandem repeat of 50-60 copies of the HCMV-IE gene. With one layer of DNP-TSA plus deposition and subsequent detection with a fluorochrome-conjugated antibody, four HRP-ODNs were needed to detect the HCMV-IE integration site. When employing two TSA amplification rounds, one HRP-ODN was sufficient for obtaining a strong signal of the integrated gene cluster, indicating that 50-60 HRP molecules can be detected with ease. In addition to DNA FISH, we report here the first use of HRP-ODN probes for mRNA detection by FISH. A single HRP-ODN and one DNP-TSA plus step resulted in clear visualization of the HCMV-IE gene transcripts in rat 9G cells induced for HCMV-IE expression by cycloheximide. Two TSA detection steps enhanced signal intensities even further. Parallel experiments with hapten-labeled ODN and cDNA probes and conventional detection methods illustrated the superiority of the HRP-ODN/TSA approach in DNA and RNA FISH.