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Cytogenetics - Coggle Diagram

- - - - Joined at centromere
    - - Required for spindle fibre attachment during mitosis
    - - Ends of chromosomes - also repetitive DNA
  - - - Metaphase chromosomes are prepared from human blood by
        
        Culturing in the presence of growth stimulators
        
        Blocking at metaphase
        
        Colchicine inhibits microtubule polymerisation by binding tubulin
        
        Staining and analysing metaphase spread
    - - Staining with DNA stains to generate reproducible banding pattern
      - Light + dark regions due to differences in sequences - AT rich = dark
      - G-banding (giemsa stain)
      - R-banding (reverse stain)
      - Fluorescent stain
  - - - When non-metacentric, the short arm is p and the long arm is q
      - Metacentric
        
        Near the middle
      - Subcentric
        
        To one side
      - Acrocentric
        
        Near one end
      - Telocentric
        
        At one end
      - Centromeric index
        
        The ratio of the short arm of the chromosome to that of the total chromosome
        
        Usually expressed as a %
  - - - Numbered from centromere to telomere
      - Centromere is 10 (one, zero)
      - 1p31.1
        
        Chromosome 1, short arm, region 3, band 1, sub-band 1
      - Different degrees of DNA packaging throughout metaphase
        
        Provides different resolutions of banding pattern in each chromosome 1 seen from different cells
        
        Different banding resolutions can resolve bands, sub-bands + sub-sub-bands
    - - Replicate early in S phase
      - Less condensed chromatin
      - Transcriptionally active
      - GC rich
    - - Replicate late
      - Contain condensed chromatin
      - AT rich
    - - DNA + protein cap
      - Ensures replication to tip
      - Tether to nuclear membrane
    - - Joins sister chromatids
      - Essential for chromosome segregation at cell division
      - 100s Kbs of repetitive DNA
        
        Some chromosome specific
        
        Some non-specific
  - - - 46, XY, del(1)(p21)
    - - 46, XX, t(2;4)(p12;q13)
- - - - Describing chromosomes in order to identify abnormalities
      - Karyotype
        
        Number, size, features etc.
    - - Types of chromosomal abnormalities
        
        Loss or gain of chromosome (monosomy, trisomy)
        
        Loss or gain of entire genome (monoploidy, polyploidy)
        
        Rearrangements
        
        Balanced (inversions, reciprocal + Robertsonian translocations)
        
        Unbalanced (Deletions, insertions, isochromosomes, ring chromosomes)
- - - - SKY + M-FISH permit simultaneous visualisation of all human chromosomes in diff colours
        
        Generation of SKY probes for whole chromosome analysis
        
        Pools of individual chromosomes isolated by flow cytometry or fluorescent activated cell sorter
        
        DNA isolated from from chromosome pools amplified by PCR + each is labelled with a different coloured fluorophore
        
        Labelled DNA probes combined + hybridised to chromosome spreads
        
        Labelled chromosome spreads are analysed by fluorescence microscopy either manually or via automated systems
        
        Chromosome-specific probe pools are generated from flow-sorted chromosomes then amplified = fluorescently labelled PCR
        
        Probes are made from individual chromosomes + given a colour by blending 5 fluorophores
    - - Identify those regions in metaphase spreads (or on interphase nuclei)
        
        Visualised with fluorescent tagged Abs
    - - Key point
        
        Single stranded DNA acts as specific probe + you can see it glow under microscope
      - Digoxygenin - small molecule recognised by an Ab which allows signal amplification by coupling lots of FITC
      - DNA is copied from target area of chromosome (PCR or cloned)
        
        When heated the double strand separates leaving either strand to hybridise back together or stick to area of chromosome identical
        
        When copied by DNApol, some of the dGTPs have FITC stuck (copied DNA contains fluorescent structure)
      - Fluorescein-12-dGTP
        
        Fluorescein isothiocyanate (FITC) attached to standard dGTP
    - - Level needed to detect micro-deletion syndromes
        
        Velocardiofacial syndrome/DiGeorge
        
        Angelman syndrome (maternal)
        
        Prader-Willi (paternal)
        
        Williams syndrome