Chromosome banding Flashcards
What is the most commonly used banding method in North America?
G-banding
What are the dark bands created by G-banding called? What do they contain?
G-bands. They have a low G/C content and a low gene density, and are more heterochromatic
What are the light bands created by G-banding called? What do they contain?
G-negative bands. They have a high G/C content and a high gene density, and are more euchromatic
What is the 400 band resolution?
The number of bands you get if you G-band chromosomes at the height of metaphase, when the chromosomes are the most condensed
How do you increase resolution of banding?
Harvest cells and stain earlier to get less condensation
Why do we want higher resolution when karyotyping?
Makes it easier to detect translocations
What is the 550 band resolution?
The number of bands you get if you G-band chromosomes in early metaphase, when the chromosomes are less condensed
What is the 800 band resolution?
The number of bands you get if you G-band chromosomes in late prophase, when the chromosomes are past the spaghetti phase but are less condensed
How do you do high resolution banding?
Add a bunch of dTTP to mess with the nucleotide balance and stall the cells in S phase and synchronize them. Then time your harvest to view the chromosomes in late prophase
What is the standard notation for nomenclature of chromosome bands?
Chromosome arm (p or q), region, band, period, sub-band, sub-sub-band. Each number is read separately
What defines a region on a chromosome? Which direction does the numbering go?
Regions are defined by distinct morphological features like centromeres, constrictions, and telomeres. The numbering starts at 1 near the centromere and goes out towards the telomere
Is region p1 more proximal or distal to the centromere than region p2?
Proximal
Is band p15 more proximal or distal to the centromere than band p12?
Distal
Is sub-band p13.4 more proximal or distal to the centromere than sub-band p13.1?
Distal
Is sub-sub-band p11.43 more proximal or distal to the centromere than sub-sub-band p11.45?
Proximal
Where does the “G” in G-banding come from?
Giemsa, the stain
Why do we do a controlled trypsin digestion before G-banding?
Without it, the entire chromosome stains dark and we don’t get any bands
What is the advantage of G-banding over Caspersson’s Q-banding method?
Get the same pattern, but the staining is permanent. No need to worry about quenching
What is the name for a chromosome that has undergone a translocation?
A derivative chromosome
What is R-banding?
Reverse banding. Staining procedure that creates the opposite pattern as G-banding
What is the difference in the procedure between G-banding and R-banding?
R-banding adds one more step, which is heat denaturation in saline before the trypsin digestion and giemsa staining
Why does the heat denaturation step in R-banding create the opposite banding pattern as G-banding?
The A/T rich regions of the G bands gets denatured and can’t hold the stain, so they stay light coloured and the G/C rich G-negative bands stay together and get stained instead
What is chromomycin A3?
Fluorescent dye that binds to G/C rich regions and produces the same banding pattern as R-banding
What is acridine orange staining?
A modified version of the R-banding protocol. Acridine orange is an intercalating agent that fluoresces orange when bound to ssDNA and fluoresces yellow/green when bound to dsDNA
What are the two bands we get with acridine orange staining?
Orange bands are the A/T rich G-bands and are from acridine orange binding to ssDNA that was heat denatured
Yellow/green bands are the G/C rich R-bands that are from acridine orange binding to dsDNA
What are T-bands?
A subset of highly intense R-bands near the telomere
How do you do T-banding?
Use much harsher heat denaturation
What are C-bands?
A subset of G-bands localized to constitutive heterochromatin
How do you do C-banding?
BaOH treatment instead of heat denaturation before staining
What is NOR staining?
Staining rRNA genes in the stalk of acrocentric chromosomes - nucleolar organizing regions
What are 2 ways to do NOR staining?
- Silver nitrate
2. Fluorescent probes for rRNA genes and in situ hybridization
What are fragile sites?
Non-staining regions at characteristic sites on several chromosomes
How do you view fragile sites?
Treat the cells with something that inhibits DNA synthesis, like having high nucleotide concentrations
