Chromosome Structure and Banding Flashcards
What is a chromosome band?
Part of a chromosome that can be distinguished from adjacent segments by appearing lighter or darker by one or more techniques.
What are the two principal groups of banding?
- those that produce thin, alternating bands along the entire length of the chromosomes (G,Q and R)
- those that stain only a specific band or region of some or all of the chromosomes( C,T,NOR, DAPI/DA)
What is the most widely used method for staining mammalian chromosomes
Geimsa banding (G-banding)
Why is G-banding so widely used?
relatively simple technique, reliable and reproducible and does not require a fluorescent microscope for visualisation.
What is the max resolution for G-banding?
3~5Mb.
What is used to produce a G-banding evaluation score?
Specific G-bands at any given banding resolution (<300 bands to >900 bands) are used to produce a G-banding evaluation score which allows the definition of the lowest standard acceptable for a given reason for referral in chromosome analysis according to the best practice guidelines
What phase must chromosomes be in for G-banding?
G-banding is performed by treating aged metaphase chromosome preparations (from 30-90 mins at 90°C to 3-5 days at room temperature) with a protease (dilute trypsin) or with hot 2x SSC, and then staining with Giemsa stain or similar chromatin stain (such as Leishman’s stain).
What causes the purple colour produced in G-banding?
The purple colour is produced when Giemsa stains chromatin arises from the precipitation of hydrophobic compounds
How are G-light and G-dark bands produced?
After trypsin treatment, the application of Giemsa stain produces a reproducible pattern of G-dark and G-light bands.
Trypsin, acts upon the chromatin scaffold leading to suggestions that the G-banding pattern is produced by direct interaction between Giemsa and these proteins.
Key features of G-dark
Heterochromatin
AT-rich
Gene-poor
Low histone acetylation
Long Intermediate Nuclear Elements
Key features of G-light
Euchromatin
GC-rich
Gene-rich
High histone acetylation
Short Intermediate Nuclear Elements
Which staining method produces a complementary banding pattern to G-banding?
R (reverse) banding produces a complementary but not mirror image, banding pattern to that of G-banding (dark G-bands are light by R-banding)
What is an advantage of R-banding over G-banding?
Telomeric regions are generally stained more darkly, allowing for improved visualisation if telomeres are involved in aberrations
R-banding standard protocol?
The generalised protocol for R-banding involves incubating chromosomes in a hot (85-90°C) phosphate buffer followed by Giemsa staining.
What was the first staining technique used on human chromosomes?
Q- Banding
What is Q-banding?
Q-banding is a fluorescent technique. Quinacrine dihydrochloride
binds to DNA and produces distinct banding patterns of bright and dull fluorescence when excited with the proper wavelength of light. Because adjacent A-T pairs are necessary to create binding sites, the brightly fluorescing regions are A-T rich.
How does a Q-banding pattern, differ from a G-banding pattern?
- the large polymorphic pericentromeric regions of chromosomes 1 and 16, and the distal long arm of the Y fluoresce brightly
- the distal long arm of the Y chromosome is the most fluorescent site in the human genome.
- Q-band polymorphic regions at the centromeres of chromosomes 3 and 4 that cannot be appreciated with G-banding.
What is an advantage of Q-banding over G-banding?
Q-banding can be useful to confirm the presence of Y material or when studying the known polymorphic regions.
What is a disadvantage of Q-banding over G-banding?
Quinacrine dye requires fluorescence microscopy for visualisation and the staining is prone to fading with time.
Advantages and disadvantages of DAPI
Advantages: Only staining technique that highlights 15p, quick protocol.
Disadvantages: Fades quickly therefore no permanent preparation.
What is DAPI?
a fluorescent dye with DNA affinity for A + T-specific binding. Chromosomes stained by DAPI alone exhibit Q-band like fluorescence along the length of the chromosomes, but with prominent staining of the heterochromatic regions of chromosomes 1 and 16.
Describe some uses of DAPI
Staining pattern highlights the following bright blue: 1qh, 9qh, 16qh,Yqh, distal short arm of chromosome 15. The rest of the chromosomes stain pale blue.Polymorphisms- as for C banding; 15p polymorphisms and identification of markers that satellited, e.g. dic(15) or idic(15).
What is Bloom syndrome?
Rare, autosomal recessive disease ass. with primordial dwarfism, sun-sensitive skin lesions, characteristic facies and immunodeficiency, azoospermia in males /POF in females.
Dysfunctional BLM no longer prevents SCEs after DNA damage leading to hyper-recombinability and high spontaneous SCEs frequently observed up to 10x more than normal individuals.
What are sister chromatid exchanges (SCEs)?
SCEs: occur spontaneously, distribution non-random. They are a reciprocal exchange of genetic material from two identical chromatids following their duplication in S phase of the cell cycle. Normal human cells have 5-8 SCEs per mitosis.
What is Nucleolar organiser region (NOR) staining?
Nucleolar organizser regions are known to contain the genes for 18S and 28S rRNA (located on the short arms of acrocentric chromosomes in humans). Regions in which these genes are actively transcribed can be stained using silver nitrate, the remaining complement stains lightly brown/yellow.
Only technique for staining satellite stalks, much cheaper than FISH
Other types of banding (place holder)
T-Banding/CT banding
Cd Staining ( Centromeric Dot or Kinetochore Staining)
G-11 Banding (Geimsa at pH 11 )
DAPI and DAPI-Distamycin A staining
