FluorescenceinsituHybridization-Universityof.pptVIP

Fluorescence in situ Hybridization Fluorescence in situ Hybridization (FISH) FISH - a process which vividly paints chromosomes or portions of chromosomes with fluorescent molecules Opening picture - Human M-phase spread using DAPI stain Fluorescence in situ Hybridization (FISH) Identifies chromosomal abnormalities Aids in gene mapping, toxicological studies, analysis of chromosome structural aberrations, and ploidy determination Fluorescence in situ Hybridization (FISH) Used to identify the presence and location of a region of DNA or RNA within morphologically preserved chromosome preparations, fixed cells or tissue sections Fluorescence in situ Hybridization (FISH) This means you can view a segment or entire chromosome with your own eyes Was often used during M phase but is now used on I phase chromosomes as well Fluorescence in situ Hybridization (FISH) Advantage: less labor-intensive method for confirming the presence of a DNA segment within an entire genome than other conventional methods like Southern blotting FISH Procedure Denature the chromosomes Denature the probe Hybridization Fluorescence staining Examine slides or store in the dark FISH Procedure FISH Uses Detection of high concentrations of base pairs Eg: Mouse metaphase preparation stained with DAPI (a non-specific DNA binding dye with high affinity for A-T bonds) FISH Uses FISH Uses Centromere regions stained brighter - means they are rich in A-T bonds Also used in germ cell or prenatal diagnosis of conditions such as aneuploidies FISH and Telomeres Telomeric probes define the terminal boundaries of chromosomes (5’ and 3’ ends) Used in research of chromosomal rearrangements and deletions related to cell aging or other genetic abnormalities FISH and Telomeres Special telomeric probes specific to individual chromosomes have been designed Probe is based on the TTAGGG repeat present on all human telomeres FISH and Telomeres FISH and Telomeres Application in cytogenetics - can detect submicroscopic