W Deng1, J N Lucas. 1. Lawrence Livermore National Laboratory, University of California, Livermore 94551, USA.
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 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. 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 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. 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.
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 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. 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 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. 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.
Authors: Wen Deng; Pei Shin Pang; Chi Man Tsang; Pok Man Hau; Yim Ling Yip; Annie L M Cheung; Sai Wah Tsao Journal: PLoS One Date: 2012-06-25 Impact factor: 3.240