Chromosome

• Human genome

  All of the genes found in a single individual
• The human genome consists of 2.9 billion base pairs of DNA organized in 23 chromosomes

  
• Diploid organisms have 46 chromosomes
• Mutation


  A transmissible (inheritable) change in DNA sequence
• Polymorphism


  A difference in DNA sequence found in 1% to 2% more of a given population
• Examples of polymorphisms

  • ABO blood groups
  • Major histocompatibility complex
  • Polymorphisms used for human ID and paternity
• Mutation

  A change in the order or sequence of nucleotides in DNA found in less than 1% to 2% of the given population
• Balanced polymorphism


  Maintained in a population through a balance of positive and negative phenotype
• Example of balanced polymorphism

  • Sickle cell anemia - Abnormal RBC resulting into resistance to infection by Plasmodium species (malaria)
• Classification of mutations

  • Gene mutations - Affect single genes & are often but not always, small changes in the DNA sequence
  • Chromosome mutations - Affect the structures of entire chromosomes
  • Genome mutations - Changes in the number of chromosomes (aneuploidy)
• Chromosome behavior

  • Dependent on chromosome structure and DNA sequences
  • Position effect - Chromosome topology affects gene activity. Highly compacted DNA = less available for RNA transcription
• Chromosome visualization techniques


  • Conventional cytological stains: Feulgen, Wright and hematoxylin
  • Fluorescent dyes: quinacrine and quinacrine mustard - Pattern: Q banding
  • Chemical dye: Giemsa stain - Pattern: G bands
  • Harsher treatment of chromosomes (87°C for 10 min, then cooling to 70°C) before Giemsa staining - Pattern: R bands
  • Alkali treatment (centromere staining) - Pattern C bands
  • Nucleolar organizing region (NOR) staining - Silver nitrate - stain specifically the constricted regions, or stalks, on the acrocentric chromosomes
  • 4'6-diamidino-2-phenylindole (DAPI): binds to the surface grooves of dsDNA - Fluoresces blue under UV light (353-nm wavelength) - Visualization of chromosomes and whole nuclei
• Chromosome banding

  • Facilitates the detection of deletions, insertions, inversions, and other abnormalities
  • Identifies distinct chromosomal locations

  
• Methods for detecting genome and chromosomal mutations

  • Flow cytometry - Indirect method of detecting genome mutations, or aneuploidy
  • Karyotyping - Direct method of detecting genome mutations, or aneuploidy - Observation of metaphase chromosome structure by arranging them according to size
• Chromosomal mutations detected by karyotyping

  • Translocation - Exchange of genetic material between chromosomes
  • Deletion - Loss of chromosomal material
  • Insertion - Gain of chromosomal material
  • Inversions - Result from excision, flipping, and reconnecting chromosomal material within the same chromosome
  • Isochromosome - Metacentric chromosome resulting from transverse splitting of centromere during cell division
  • Ring chromosome - Results from deletion of genetic regions from ends of the chromosome and a joining of the ends to form a ring
  • Derivative chromosome - Translocated/otherwise rearranged parts from 2 or more unidentified chromosomes joined to a normal chromosomal
• The first chromosome mutations associated with human disease were visualized in the 1960s in leukemia cells
• Terms used in expressing karyotypes

  • +: Gain
  • : Loss

  del: Deletion

  der: Derivative chromosome

  dup: Duplication

  ins: Insertion

  inv: Inversion

  i, iso: Isochromosome

  mat: Material origin

  pat: Paternal origin

  1. Ring chromosome
  2. Translocation

  tel: Telomere (end of chromosome arm)
• Fluorescence in situ hybridization (FISH) techniques

  • Interphase FISH - Commonly used to study prenatal samples, tumors, & hematological malignancies

  Metaphase FISH - Allows analysis of small regions not visible by regular chromosome banding

  Multicolor FISH (QMFISH or M-FISH) - Simultaneous use of combinations of different locus-specific probes & chromosome paints

  
• Steps in FISH

  • Preparation of sample - Permeabilize the cells, fix with formaldehyde, dehydrate in ethanol, denature

  Quality of the probe - Signal should be bright, specific to the target, and low background

  Denaturation - Both probe & target must be denatured prior to hybridization

  Washing - Rinsing off of the unbound probe

  Microscopic analysis - Observe under fluorescent microscope, adequate number of cells must be visible

  
• Preparation of chromosomes for metaphase FISH


  • Culture of cells for 72 hours

  45 mins before harvesting, colcemid is added

  Cells are suspended in a hypotonic medium (0.075 M KCH & fixed with methanol/acetic acid (3:1)

  Fixed cell suspension is applied to an inclined slide & allowed to dry

  2nd treatment w/ 70% acetic acid

  Chromosome should appear well separated w/ sharp borders & cytoplasm should not be visible

  
• Comparative Genome Hybridization (CGH)

  Detection of intrachromosomal amplifications/deletions - Test DNA is isolated & labeled along with a reference DNA - Fluorescent labels: Cy3 (green), Cy5 (red), Cy3.5 (red-orange) - Labeling is achieved by nick translation/primer extension
• Causes of chromosome abnormalities

  • Error in cell division - can result in cells w/ too few or too many copies of a chromosome

  Maternal age - chromosomal errors can appear in eggs as women age

  Environmental conditions - conclusive evidence is currently lacking