Fluorescence in situ Hybridization Fluorescence in situ Hybridization
Fluorescence in situ Hybridization
Fluorescence in situ Hybridization (FISH) w FISH - a process which vividly paints chromosomes or portions of chromosomes with fluorescent molecules w Opening picture - Human Mphase spread using DAPI stain
Fluorescence in situ Hybridization (FISH) w Identifies chromosomal abnormalities w Aids in gene mapping, toxicological studies, analysis of chromosome structural aberrations, and ploidy determination
Fluorescence in situ Hybridization (FISH) w 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) w This means you can view a segment or entire chromosome with your own eyes w Was often used during M phase but is now used on I phase chromosomes as well
Fluorescence in situ Hybridization (FISH) w 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 w Denature the chromosomes w Denature the probe w Hybridization w Fluorescence staining w Examine slides or store in the dark
FISH Procedure
FISH Uses w Detection of high concentrations of base pairs w Eg: Mouse metaphase preparation stained with DAPI (a non-specific DNA binding dye with high affinity for A-T bonds)
FISH Uses
FISH Uses w Centromere regions stained brighter - means they are rich in A-T bonds w Also used in germ cell or prenatal diagnosis of conditions such as aneuploidies
FISH and Telomeres w Telomeric probes define the terminal boundaries of chromosomes (5’ and 3’ ends) w Used in research of chromosomal rearrangements and deletions related to cell aging or other genetic abnormalities
FISH and Telomeres w Special telomeric probes specific to individual chromosomes have been designed w Probe is based on the TTAGGG repeat present on all human telomeres
FISH and Telomeres
FISH and Telomeres w Application in cytogenetics - can detect submicroscopic deletions and cryptic translocations of genes associated with unexplained mental retardation and miscarriages
FISH and Telomeres - Medical from Tel. Vysion DNA website w “A translocation between chromosomes 12 and 21 with breakpoints at bands 12 p 13 (TEL)[telomere] and 21 q 22 (AML 1) occurs in at least 25 percent of childhood B-cell acute lymphocytic leukemia”
FISH and Telomeres - Medical w The “LS 1 TEL/AML 1 ES Dual. Color Translocation Probe” is designed to detect the TEL and AML 1 gene fusion w This fusion is undetectable by standard cytogenetics, but can be seen with FISH
FISH - Medical w FISH can be used in the study of transgenic animals (eg: Polly) w Selective markers show if the human DNA was inserted successfully and pinpoint where the human DNA is w Transgenic Mouse
FISH - Medical
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