Using Fluroscence In Situ Hybridization (FISH) Technique For Evaluation Of Frequency Of Chromosomal Rearrangements In Ataxia Telangiectasia Lymphoblastoid Cells



Introduction: Fluorscence in situ hybridization (FISH) enables specific detection of unique sequences of varying length, chromosomal regions or entire chromosomes within metaphase or interphase cells. Recent developments in this technology permit the rapid mapping and ordering of DNA fragments on single metaphase chromosome bands. The technique of FISH incorporates several stages including: probe preparation and labeling, hybridization of probe with chromosome preparation and detection of signal , and imaging. Using this technique chromosomal alterations in ataxia telangiectasia lymphoblastoid cells which are highly sensitive to clastogenic and mutagenic effects of chemnical and physical agents were investigated. Materils and Methods: Normal and A-T cells were grown in RPMI – 1640 medium and irradiated at G2 and G1 phases of the cell cycle. After democolcine treatment and metaphase chromosome preparation, slides were prepared and FISH is performed for all samples using whole chromosome probe for chromosomes 5, 1, 4, 7 and 14. Fifty mitoses were analyzed for each sample by Zeiss fluorescent microscope attached to a computerized programme. Results: Analysis of normal and A – T mitoses before and after irradiation revealed a very low frequency of chromosomal abnormalities, for specific chromosomes 5 painted in this study. However, more aberration were found in A-T cells of various chromosomal rearrangements such as translocation, trisomy, Robertsonian translocation and chromatid type breaks. Conclusion: In routine cytogenetic methodologies visualisation of various chromosomal rearrangements is not always possible in a single preparation. Although detection sensitivity of FISH is limited to the specific chromosomes, but as seen for A – T cells, FISH can be considered as one of the most effective method for visualization of various chromosomal alterations. This technique can have wide application not only in human genome mapping and the genome of other organisms, but also in clinical cytogenetics, somatic cell genetics, cancer diagnosis and gene expression studies.