Cytogenetics

1
Cytogenetics

• AHMG Fall 2004
• Juniata College

2
History

• 1890-1920 techniques of plant and insect cell
  staining applied to human cells
• 1923 XX/XY was postulated to be the
  sex-determining mechanism
• 1956 46 chromosomes in human cells
• First karyotype
• 1959 Lejeune Down Syndrome (extra small
  chr.), Turner Syndrome, Kleinfelter Syndrome
  other trisomies visualized
• 1960 phytohemoglutanin (PHA)stimulates blood
  cells to divide
• When they divide, they condense to we can see
  them better

3
History

• 1970 chromosome banding
• 1977 high resolution banding
• 1986 fluorescence in situ hybridization (FISH)

4
Cell culture

• Types of cells that can be cultured for
  chromosome analysis
• Peripheral blood lymphocytes
• Amniocytes
• Chorionic villi
• Bone marrow
• Fibroblasts
• Tumors

5
Preparing a karyotype

• Grow cells in culture (mitogenic stimulation with
  PHA)
• Disrupt spindle formation with colemid
• Place in a hypotonic solution (lyses cells and
  swells the nucleus)
• Fix cells (kills them and partially extracts
  histones)
• Use methanol/acetic acid
• Drop fixed nuclei onto slides
• Stain (usually G-banding)
• Analyze with light microscopy and photograph for
  karyotype preparation

6
G-banded chromosome 21at different resolutions
beside ideograms
7
Cytogenetics

• About 400 metaphase bands (haploid)
• About 6 million bp per band
• About 80-100 genes per band
• Can see only gross changes (about 10 genes or
  more), not a single gene deletion
• G-banding dark bands are AT rich, light bands
  are GC rich
• Centromeres are AT rich (stain darkly)
• Light regions are generally more rich with coding
  sequences (promoters are GC rich)

8
(No Transcript)
9
(No Transcript)
10
(No Transcript)
11
Other cytogenetic techniques

• Fluorescence in situ hybridization (FISH)
• Spectral karyotyping
• Comparative genome hybridization (CGH)

12
FISH
13
Spectral karyotyping