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Fluorescence in situ hybridization (FISH) and the various types of FISH.

Fluorescence in situ hybridization (FISH) and the various types of FISH. Régen DROUIN, Geneticist MD, PhD, FACMG, FCCMG. Department of Medical Genetics, CHUS & Department de Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada. Cytogenetics:. - Chromosome Cytogenetics

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Fluorescence in situ hybridization (FISH) and the various types of FISH.

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  1. Fluorescence in situ hybridization (FISH) and the various types of FISH. Régen DROUIN, Geneticist MD, PhD, FACMG, FCCMG Department of Medical Genetics, CHUS & Department de Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

  2. Cytogenetics: - ChromosomeCytogenetics - InterphaseCytogenetics - Conventional Cytogenetics - Molecular Cytogenetics Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

  3. Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

  4. Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

  5. Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

  6. Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

  7. Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

  8. Molecular Cytogenetic Techniques available: • FISH (Fluorescence In Situ Hybridization) • & variants: Q-FISH, express FISH, etc. • - PRINS (PRimed IN Situ labeling) • M-FISH (Multicolor-FISH) or SKY • (spectral Karyotype) • Band-FISH • - CGH (Comparative Genomic Hybridization) Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

  9. Applications of Molecular cytogenetics • Chromosome Identification •  Aneuploidy Detection •  Centromere Analysis •  Identification of Marker Chromosome •  Whole Chromosome Analysis (chromosomepainting) •  Analysis of chromosome translocation • Detection of unique sequence (single-copy sequence) • Microdeletion investigation • Analysis of gene amplification Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

  10. FISH Fluorescence in situ hybridization Hybridation in situ avec visualisation en fluorescence Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

  11. GCAATCGCCAATTATTCCAGGACTGGG CGTTAGCGGTTAATAAGCTCCTGACCC Double-strand DNA Denatured DNA Single-strand DNA Hybridization

  12. Fluorescence In Situ Hybridization and Molecular Cytogenetics 1. Introduction History: ISH: John (1969), radioisotope probes hybridized to cell preparations and using autoradiography to detect the hybridization of the probes. FISH: Pinkel (1986), Immunofluorescence technique - safety, rapidity, low background… Afterwards, so many techniques derived from FISH have been developed, e.g. CGH, SKY, M-FISH, fiber- FISH, PRINS… Application: Gene mapping, detecting chromosome and gene changes… Metaphase chromosomes---from all types of cytogenetic preparations; Interphase cells---from above plus cytomorphological preparations; Tissue sections---from tissue biopsy slides Principles: See Fig. Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

  13. 2. Fluorescence and fluorescence microscope Electromagnetic spectrum: UV (<400 nm, invisible) violet, blue, green, yellow, orange and red (400-700 nm, visible) infrared (>700 nm, invisible). Energy increase with the wave length decrease. Fluorescence: Fluorescence, Long wave light UV or short wave light Some fluorochromes (dyes) FITC (Fluorescein isothiocyanate) Rhodamine Texas red DAPI (4’6-diamidino-2-phenylindole) PI (Propidium Iodide) heat electron Excitation (absorb energy) Illumination (release) Fig.: The principle of fluorochromes Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

  14. Fluorescence microscope: Light source: High-pressure mercury vapor lamps, tungsten-halogen lamps, or xenon lamps. Eye or Camera Filters: 1. Exciting filter, to let a certain wave length of light pass so that can excite the given fluorochrome carried on sample. 2. Barrier filter, to allow the visible light pass so that the fluorescence can be seen by eyes or the image can be captured. Transmitted and epi-illumination Special requirements: No auto-fluorescence in any part of light path except for samples Specimen on microscope slide

  15. 3. Probes---a specific DNA fragment, usually 1 to 100 kb length, complementary to the chromosome site that we are interested in. Probe Labeling: a). Indirect labeling, need antibodies to complete FISH procedure Haptens---Biotin-dUTP, digoxigenin-dUTP, b). Direct labeling, the probe directly labeled with fluorochromes such as SpectralGreen and SpectralOrange. One-step hybridization. Labeling techniques: a). Nick translation b). Random priming c). PCR (Polymerase chain reaction) DNA Polymerase adds dNTP, labeled dUTP at 3’ and remove dNTP at 5’ DNase I makes nicks 5‘ 3‘ 5‘ 3‘ ++※+++ +※++※+ 3‘ 3‘ 5‘ 5‘ +※++※ Fig.: Principle of Nick Translation Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

  16. Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

  17. 4. Hybridization a). Denaturing of DNA probes b). Denaturing of DNA template (chromosome) c). Annealing (renature, hybridization) d). Post-hybridization wash, stringency control e). Counterstaining Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

  18. 4. Hybridization a). Denaturing of DNA probes b). Denaturing of DNA template (chromosome) c). Annealing (renature, hybridization) d). Post-hybridization wash, stringency control e). Counterstaining Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

  19. FISH targets : - Metaphase Chromosomes - Interphase Nuclei - Fixed Tissues - Cells in culture Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

  20. Chromatin fiber of packed nucleosomes DNA double helix Extended section of chromosome ‘String-of-beads’ form of chromatin Condensed section of chromosome 10nm 700nm 30nm 300nm 1 400nm 2 nm DNA Condensation and fiber-FISH

  21. A good FISH method should have: - An extremely high specificity (extremely low background) - A good sensitivity (good hybridization efficiency) - Unambiguous recognition of the hybridization signal Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

  22. Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

  23. Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

  24. Genetic diseases identified using molecular cytogenetics • Prader-Willi Syndrome • Angelman Syndrome • Miller-Dieker Syndrome • Williams Syndrome de Williams • DiGeorge and velo-cardio-facial Syndromes • Wolf-Hirschhorn Syndrome • Smith-Magenis Syndrome • Kallmann Syndrome • etc... Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

  25. Di George normal Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

  26. Di George normal Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

  27. Deletion of one Di George locus Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

  28. Prader-Willi Syndrome (del.15q11-q13 pat ou DUP mat)  Mental Retardation and behavior problems  Deglutition problems and hypotonic newborn  Bulimia presented by older children (obesity)  Hypogonadism and incomplete puberty  Acromicry Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

  29. Angelman Syndrome (del.15q11-q13 mat ou DUP pat)  Severe Mental Retardation  Episodes of uncontrolled laughing  Characteristic Facial Dysmorphism (low jaw and protruding tongue)  Special behavior with disorganized movements (ataxic gait)  Convulsions Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

  30. PWS SNRPN 15q11q13 15q22 control PML SNRPN 15q11q13 15q22 control PML Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

  31. PWS 15q22 control PML SNRPN 15q11q13 15q22 control PML del (15) (q11q13) Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

  32. DEFINITION OF CRYPTIC CHROMOSOME REARRANGEMENTS These are chromosomal anomalies not visible using standard high resolution cytogenetic technique (  550 bands per haploid genome). These anomalies are detectable only when using molecular cytogenetic techniques. Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

  33. Pedigree Deceased MDS ? Normal Normal Current pregnancy (MDS) Miller-Dieker Syndrome Spontaneous Abortion Deceased MDS Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

  34. Father Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

  35. Father Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

  36. FISH (Oncor) MDS Probes 17p13.3 et RARA CR 17q21.1 16 17 17 Father of the propositus Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

  37. PRINS PRimed IN Situ labeling Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

  38. Telomere Simple DNA sequence (T2AG3) tandemly repeated, of variable length, located at the extremities of the chromosomes. Telomeres are essential elements that protect the extremities of the chromosomes from degradation and ligation. Shortening Elongation Equilibrium Incomplete Replication Addition of repetitions T2AG3 by the telomerase Nuclease Activity Senescence

  39. TTAGGG AATCCC TTAGGG AATCCC TTAGGG AATCCC Telomeres • Specialized structures made of DNA and PROTEINS • Repeated DNA sequence: 2 à 15 kb • Maintain the chromosome stability • Around 30 to 120 bp are lost per somatic cell division • Too short : cellular senescence and genetic instability Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

  40. Measurement of telomeres Average length of telomeres : • Measurement of terminal restriction fragments. • Digestion using restriction enzymes of purified DNA • Visualization and measurements of telomeric fragments by Southern blot • Cleavage of telomeres at variable distance • No information individual telomeres Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

  41. Measurement of telomeres Length of individual telomeres : Quantitative FISH (Q-FISH) Hybridization telomeric PNA probes Measurements of the signal intensity Length Profil of individual telomeres Variation of hybridization efficiency ??? Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

  42. 2.9 m 100kb/29 m 100kb/19.5 m 2.1 m 100kb/14.1 m 1.5 m 1. Measurement of 1q telomere

  43. Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

  44. Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

  45. Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

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