DNA is associated with histones. Nucleosomes: 8 histones associated with 147bp of DNA and 50 bp of linker DNA. Linker DNA + H1 histone. Histone octamer + 147bp of DNA.
DNA Packaging:
Euchromatin: Less compacted DNA, transcriptionally active.
Metaphase cells are required to prepare a standard karyotype.
Cells are cultured in the presence of a mitogen (phytohemagglutinin).
Treated with colchicine or demecolcine (colcemid) to disrupt mitotic spindle and prevent completion of mitosis.
Cells treated with a hypotonic solution.
Cells are fixed, dropped onto a microscope slide and dried.
Slides are stained.
Chromosome Banding Techniques
Methods resulting in bands distributed along the length of the whole chromosome, such as G-, Q- and R-bands.
G banding (Giemsa & trypsin).
Q banding (quinacrine).
R banding (reverse banding).
C banding (centromere staining).
Bright field G bands:
Most commonly used.
The dark regions tend to be heterochromatic, the bright regions tend to be euchromatic.
C-bands shows the centromeric region of chromosomes.
Resolution in the Human Karyotype
Total number of bands depends on how condensed the chromosomes are, and at what stage of mitosis.
A 350-band resolution corresponds to chromosomes late in metaphase.
Each sub-band is placed behind a decimal point following the first band designation.
Methods Staining Specific Bands/Structures
Fluorescence In-Situ Hybridization (FISH):
Method used to identify specific parts of a chromosome.
Accurately localises specific gene sequences.
Developed with the availability of DNA probes and non-radioactive DNA labelling procedures.
Necessary to know what to look for in order to make the appropriate probe.
Useful to find to which chromosome a certain gene belongs.
Spectral karyotyping (SKY):
Chromosome-specific probes are made by labeling DNA fragments covering each individual chromosome with a specific colored fluorescent dye.
Each individual chromosome is labeled with a specific fluorescent colour.
Simultaneous tracking of all human chromosomes.
Chromosomal translocations are easily detected.
Chromosomal microarray:
Arrays containing thousands of base-pair fragments of the human genome adhered to a microchip.
Each individual DNA fragment corresponds to a known DNA sequence, mapped to a specific chromosomal region.
Color-coded probes are used to label the patient and the normal DNA differently: in the example, red for the normal control and green for the patient.
Unaltered chromosome regions show equal binding of the green and red probes (orange/yellow colour resulting from green overlapping to red, showing co-hybridization).
Amplified and deleted chromosomal regions in the patient appear either red or green.