Improved in situ detection method for telomeric tandem repeats in metaphase spreads and interphase nuclei

Peptide nucleic acid technology (PNA) has become an extremely useful tool a nd promises to impact on molecular biology and diagnostics. These synthetic DNA analogues pair with DNA and RNA molecules according to Watson and Cric k base pairing rules. This paper describes a sensitive and quick fluorescen t in situ hybridisation (ISH) technique to determine DNA telomere repeat se quences (TTA GGG)(n) using epifluorescence microscopy. Telomeres are specia l, repeated structures at the end of each eukaryotic chromosome and serve a s protective caps to prevent DNA rearrangements and fusion of chromosomes. A model system has been developed, using stimulated peripheral blood lympho cytes, which facilitates simultaneous detection of telomeres in metaphase a s well as in interphase nuclei. A fluorescein isothiocyanate labelled FNA p robe (18 mer) directed against complementary telomeric sequences at the end of each chromosome is used. In addition, a simple, easy to perform PNA-ISH protocol is described that overcomes common hybridisation problems encount ered using DNA and RNA oligoprobes. Furthermore, the usefulness of a chromo genic immunocytochemical detection system is shown for PNA-ISH.