Combined FISH with pan-telomeric PNA and whole chromosome-specific DNA probes to detect complete and incomplete chromosomal exchanges in human lymphocytes
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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DOI：
10.1080/095530099139575
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发表时间：
1999-09-01
影响因子：
2.6
通讯作者：
Lucas, JN
中科院分区：
文献类型：
--
作者：
Deng, W;Lucas, JN
目的：将FISH技术与端粒多肽核酸和全染色体特异性DNA探针相结合，检测人淋巴细胞染色体的完全交换和不完全交换。材料和方法：采用0.9Gy低剂量率(0.019 Gyh)氚体外照射人淋巴细胞。中期铺片用核糖核酸酶处理，固定在1：3的醋酸：甲醇中，再用氯化钾、蛋白酶K进一步处理，固定在4%的多聚甲醛中。切片变性，与FITC标记的端粒PNA探针杂交1.5h，再与1、2、4号染色体橙色全染色体探针杂交过夜。杂交涂片用70%甲酰胺/20x SSC洗涤，DAPI反染色。结果：杂交后3对标记染色体和92个端粒均清晰可见。整个1、2和4号染色体被涂成橙色，所有的端粒都被染成绿色。未上色的染色体被反染成蓝色。在52h培养的人淋巴细胞经氚-β射线诱发的680条染色体畸变中，未发现端粒增加的证据。结论：通过同时检测端粒和特定的完整染色体，首次准确分析了人类淋巴细胞中染色质染色体的完整和不完全交换。
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 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.