Combined FISH with pan-telomeric PNA and whole chromosome-specific DNA probes to detect complete and incomplete chromosomal exchanges in human lymphocytes

Abstract

Purpose: To combine FISH with pan-telomeric peptide nucleic acid (PNA) and whole chromosome-specific DNA probes to detect complete and incomplete chromosome exchanges in human lymphocytes. Materials and methods: Human lymphocytes were irradiated in vitro with 0.9 Gy low dose-rate (0.019 Gy/h) tritium β-rays. Metaphase spreads were treated with RNase, fixed in 1:3 acetic acid:methanol, and then further treated with KCl, proteinase K and fixed in 4% paraformaldehyde. Slides were denatured, hybridized for 1.5 h with an FITC-labelled telomeric PNA probe, and rehybridized overnight with a spectrum-orange whole-chromosome probe specific for chromosomes 1, 2 and 4. Hybridized spreads were washed with 70% formamide/20 x SSC and counterstained with DAPI. Results: All three pairs of labelled chromosomes together with 92 telomeres were readily visible after hybridization. The whole chromosomes 1, 2 and 4 were painted orange, and all telomeres were stained green. Unpainted chromosomes were counterstained blue. In the observed 680 chromosome aberrations induced by tritium β-rays in human lymphocytes after 52 h of culture, no evidence of telomere addition was detected. Incomplete and hidden complete exchanges and terminal deletions were definitively discriminated. Conclusion: The simultaneous detection of telomeres and specific whole chromosomes allows for the first time accurate analysis of complete and incomplete chromosome exchanges involving painted chromosomes in human lymphocytes.