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.
