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

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Abstract
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. Human lymphocytes were irradiated in vitro with 0.9 Gy low dose-rate (0.019 Gy/h) tritium beta-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. 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 beta-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. 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.

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