Fusion FISH Imaging: Single-Molecule Detection of Gene Fusion Transcripts In SituReport as inadecuate




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Double-stranded DNA breaks occur on a regular basis in the human genome as a consequence of genotoxic stress and errors during replication. Usually these breaks are rapidly and faithfully repaired, but occasionally different chromosomes, or different regions of the same chromosome, are fused to each other. Some of these aberrant chromosomal translocations yield functional recombinant genes, which have been implicated as the cause of a number of lymphomas, leukemias, sarcomas, and solid tumors. Reliable methods are needed for the in situ detection of the transcripts encoded by these recombinant genes. We have developed just such a method, utilizing single-molecule fluorescence in situ hybridization sm-FISH, in which approximately 50 short fluorescent probes bind to adjacent sites on the same mRNA molecule, rendering each target mRNA molecule visible as a diffraction-limited spot in a fluorescence microscope. Utilizing this method, gene fusion transcripts are detected with two differently colored probe sets, each specific for one of the two recombinant segments of a target mRNA; enabling the fusion transcripts to be seen in the microscope as distinct spots that fluoresce in both colors. We demonstrate this method by detecting the BCR-ABL fusion transcripts that occur in chronic myeloid leukemia cells, and by detecting the EWSR1-FLI1 fusion transcripts that occur in Ewing-s sarcoma cells. This technology should pave the way for accurate in situ typing of many cancers that are associated with, or caused by, fusion transcripts.



Author: Fatu Badiane Markey, William Ruezinsky, Sanjay Tyagi, Mona Batish

Source: http://plos.srce.hr/



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