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GENETIC DIAGNOSTIC METHODS

GENETIC DIAGNOSTIC METHODS. Yrd.Doç.Dr.Ayşe Gül ZAMANİ Selçuk Üniversitesi Meram Tıp Fakültesi Tıbbi Genetik AD, KONYA. GENETIC DIAGNOSTIC METHODS. 1. Cytogenetic and Molecular Cytogenetic Diagnostic Methods I. Cytogenetic Diagnostic Methods

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GENETIC DIAGNOSTIC METHODS

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  1. GENETIC DIAGNOSTIC METHODS Yrd.Doç.Dr.Ayşe Gül ZAMANİ Selçuk Üniversitesi Meram Tıp Fakültesi Tıbbi Genetik AD, KONYA

  2. GENETIC DIAGNOSTIC METHODS 1. Cytogenetic and Molecular Cytogenetic Diagnostic Methods I. Cytogenetic Diagnostic Methods 1. Classic Cytogenetic Diagnostic Methods 2. Specialized Cytogenetic Diagnostic Methods a. Sister Chromatid Exchange( SCE) b. Micronucleus Technique(MN) c. Chromosomal Fragile Sites II. Molecular Cytogenetic Diagnostic Methods 1. Flow karyotyping 2. Flourescence in situ hybridization (FISH) 3. Fiber FISH 4. Multicolor-FISH a. Spectral karyotyping (SKY) b. Multiplex-FISH (M-FISH) c. R-FISH d. Cobra-FISH e.Comparative Cytogenetic Hybridization (CGH) 2- Molecular Genetics Diagnostic Methods I. Polymerase Chain reaction (PCR) II. Allele-specific oligonucleotid= ASO) III. DNA sequencing IV. Microarray Analysis

  3. Chromosomes  mitotic/meiotic metaphase Spontaneous dividing tissue cells Stimulated cells to divide in cultures

  4. Spontaneously dividing cells: Bone marrow Lymph nodes Solid tumors Pleural and acidic fluid cells Fetal acid cells Cystic hygroma Fetus or newborn blood

  5. Low spontaneous dividing rate Peripheral blood lymphocytes Amniocytes Chorionic villi Skin and fibroblasts

  6. CELL(TISSUE)CULTURE • Short term culture (24-72, sometimes 96 hours) Exp:Bone marrow,blood … • Long term culture(1-3 weeks) Exp: Amniocytes, skin and other tissues

  7. Culture Media and Routine Media Ingredients 1. Mediums They are prepared in balanced salt solution Hanks or HBSS→osmotic pressure and pH control Glucose→ energy for cells Mediums: TC Medium 199 Minimal Esential Medium (MEM) McCoy’s 5A Nutrient Ham’s F-10 RPMI-1640, etc Specific mediums exp: Chang Leibovitz L15

  8. Culture Media and Routine Media Ingredients 2. Antibiotics penicillin Bacteriostatic streptomycin nystatin Fungosid amphotericin 3. Serums Fetal Bovine Serum(FBS) Fetal Calf Serum (FCS) Growth factors, adhesion factors trace elements, hormones,vitamins 4. L-Glutamin

  9. Culture Media and Routine Media Ingredients 5. Mitotic stimulant = mitogen EBV(Epstein Bar Virus), Pokeweed mitogen (PWM), Phorbol esther (TPA) Phytohemagglutinin,an extract which stimulates the T-cell fraction of lymphocytes

  10. Karyotyping – cell preparation • Culture cells until sufficient mitotic activity • Add colchicine (or colcemid) to arrest in metaphase (prevents mitotic spindle fibres forming) • Add hypotonic salt solution to swell cells 0.075 M KCl ,% 0.95’lik Na Sitrat • Fix with mix of methanol;acetic acid (3 metanol:1 acetic acid) • Drop cell suspension onto the slides and allow to dry Needness for long chromosomes with none overlapping

  11. G-Banding • Most common method used • Chromosomes treated with trypsin • denatures protein • Giemsa stain • each chromosome characteristic light and dark bands • 400 bands per haploid genome • Each band corresponds to 5-10 megabases • Dark bands are gene poor

  12. Idiogram

  13. ISCN • International System for Human Cytogenetic Nomenclature • For each area of chromosome a number is given • Lowest number is closest to centromere (proximal) • Highest number is at tips to centromere (distal)

  14. High resolution Banding(HRB) • High resolution • 800 bands- prometaphase chromosome • use methotrexate and colchicine

  15. Q Banding • Used especially for Y-chromosomeabnormalities or mosaicism • Similar pattern to G banding But can detect • polymorphisms • Needs fluorescent microscope

  16. Reverse (R) Banding • Used to identify X chromosome abnormalities • Heat chromosomes before staining with Giemsa • Light and dark bands are reversed

  17. C-Banding • Used to identify centromeres / heterochromatin • Heterochromatic regions • contain repetitive sequences • highly condensed chromatin fibres

  18. NOR-Banding • Nucleolar organizer regions and seconder constrictions of acrocentric chromosomes are dyed

  19. SISTER CHROMATID EXCHANGE • To obtain SCE asymmetric incorporation of BrdU(bromodeoxyuridine) in chromosomal DNA is essential. • This achieved by incorporating BrdU, into replicating DNA for two successive cell cycles. • This results in SCE by a faintly stained chromatid containing bifiliarly BrdU-substituted DNA and a brightly stained sister chromatid unbifiliarly BrdU-substituted DNA

  20. SISTER CHROMATID EXCHANGE HARLEQUIN CHROMOSOMES

  21. MICRONUCLEUS

  22. CHROMOSOMAL FRAGILE SITES • The fragile sites are nonstaining gaps usually involving both chromatids present at the same point in the chromosome in a significant number of cells .

  23. FLOW KARYOTYPING

  24. FLOW KARYOTYPING

  25. Fluorescence In Situ Hybridisation(FISH)

  26. Whole chromosome painting probes Chromosome 8 painting probe

  27. Centromer specific probes ● chromosome 7 centromer specific signal ● chromosome 8 centromer specific signal

  28. Locus specific probes DiGeorge Syndrome ( microdeletion on 22 ) ● 22q13 specific control signal ● 22q11.2 specific DiGeorge probe SRY locus specific probe. SRY/X probe was used. ● X chromosome centromer specific probe ● SRY probe

  29. TELOMERIC PROBES

  30. Fiber FISH

  31. Spectral Karyotyping (SKY)

  32. Multiplex FISH • Metaphase spread of a lung cancer cell line after hybridization of the 5-fluorochrome M-FISH mix shown in "true-color" (a) and "classification-color" representation (b). The karyotype is shown in (c).

  33. Multiplex FISH (c).The karyotype

  34. Cobra-FISH • Overview of the 12-color image obtained using a human COBRA–FISH probe set. • Only three fluorochromes were used for ratio labeling (DEAC, Cy3 and Cy5 ). • The white arrow indicates a translocation that was not detected by conventional cytogenetic testing. • www.nature.com/.../v1/n1/full/nprot.2006.41.html

  35. R x FISH=Cross-species colour FISH • www.nature.com/.../v1/n1/full/nprot.2006.41.html

  36. Comparative Genomic Hybridization (CGH):

  37. Comparative Genomic Hybridization (CGH) DNA from Disease Tissue (TEST) DNA from Normal Tissue (CONTROL) Label Incorporating (RED Fluorescent) Label Incorporating (GREEN Fluorescent) Hybridize to Normal Metaphase Chromosome -2 0 2 (log2 ratio) Amplification Deletion

  38. Comparative Genomic Hybridization (CGH)

  39. Comparative Genomic Hybridization (CGH)

  40. Comparative Genomic Hybridization (CGH)

  41. Comparative Genomic Hybridization (CGH)

  42. Polymerase Chain Reaction(PCR)

  43. Genetical use of PCR • Oncogenetic researches • Mutation analysis (inherited disease diagnosis in patients and carriers) • Prenatal diagnosis • Pathogen microorganism detection in clinical samples • Forensic medicine (DNA fingerprinting, etc) • Probe making/cloning/gene ekspression researches • DNA sequencing • Detection of unknown DNA sequences • Ancient DNA researches • Restriction Fragment Length Polymorphism (RFLP) • Single cell analysis in IVF (in vitro fertilization) • DNA protein interaction research(footprinting)

  44. Allele-specific oligonucleotide (ASO)

  45. Allele-specific oligonucleotide (ASO)

  46. DNA Sequencing

  47. DNA Sequencing

  48. Microarray technique

  49. Microarray technique

  50. Microarray technique

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