Rapid Denaturation Improves Chromosome Morphology and Permits Multiple Hybridizations During Fluorescence in Situ Hybridization

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Abstract
Denaturation of chromosomal DNA for fluorescence in situ hybridization (FISH) is an essential step in a procedure associated with a number of variables. In our experience, shorter denaturation time in 70% formamide/2 × SSC at 72 C provides sufficient denaturation, where the hydrogen bonds are broken between the purines and pyrimidines of the double helix. This shortened exposure improves retention of morphology of human chromosomes from lymphocytes, aminocytes, fibroblasts and bone marrow, and allows the same metaphases to be denatured repeatedly and rehybridized with different probes. This approach is useful in investigations where sample volume is limited.