19.01.03 relationship between chromosome structure and banding Flashcards
g banding dye
Giemsa
Romanowsky-Giemsa effect
purple coloration of chromatin DNA, contrasting with the blue-stained RNA-containing cytoplasm and nucleoli
G-banding staining regions
heterochromatin, gene- poor, AT- Rich, low histone acetylation level, long Intermediate Nuclear Elements
G-banding method
aged metaphase chromosome preparations treated with a protease (trypsin) or with hot 2x SSC and then stained with Giemsa stain or similar chromatin stain
A chromosome band
is part of a chromosome that can be distinguished from adjacent segments by appearing lighter or darker by one or more techniques
maximum resolution of G-banding is
~3-5Mb
Differential banding pattern is due to
distribution of chromatin proteins to DNA , chromatin compaction and DNA strandedeness
Reverse Banding (R-banding) stains the
GC-rich euchromatin
Advantage of R-banding compared to G-banding
telomeric regions are generally stained more darkly, allowing for improved visualisation if telomeres are involved in aberrations
protocol for R-banding
incubating chromosomes in a hot (85-90°C) phosphate buffer followed by Giemsa staining.
Constitutive heterochromatin banding (C-banding) stains
constitutive heterochromatin, i.e. structural chromosome material comprised of repetitive and non-repetitive, satellite DNA that is mostly located at the centromeric regions of human chromosomes
Constitutive heterochromatin is
a permanent transcriptionally-repressive state, highly polymorphic. Can affect the size and the localisation of heterochromatin without observable phenotypic effect.
facultative heterochromatin is
condensed only semi-permanently via epigenetic changes which are reversible, allowing DNA transcription.
C-banding uses
to identify/confirm polymorphic variants in the lengths of the heterochromatic regions, inversions or rearrangements of chromosome 9 or other chromosomes, determining the presence of dicentric and pseudodicentric chromosomes and for studying marker chromosomes.
C-banding protocol
involves alkali treatment (usually Barium hydroxide as milder than NaOH) for DNA denaturation
Q- Banding uses
Quinacrine dihydrochloride fluorochrome.
Quinacrine dihydrochloride binds to
Adjacent A-T pairs, so A-T rich regions fluoresce brightly
Q-banding is useful for
confirming the presence of Y chromosome material (distal long arm of the Y fluoresce brightly)
Q-banding disadvantage
staining is prone to fading with time
T-Banding
Thermal treatment is used to produce staining of strictly telomeric R-bands
T-Banding is useful for
analyzing deletions or translocations involving the terminal ends of chromosomes.
CT banding
heat treatment at high pH using Barium hydroxide
Cd (Centromeric Dot or Kinetochore) Staining
produces a pair of dots at each centromere, one on each active or functional chromatid.
Difference between Cd and C banding
C-banding will stain inactive and active centromeric regions, Cd banding will only stain active
Cd banding is useful to
differentiate functional from nonfunctional centromeres and study Robertsonian translocations (centromere to centromere translocations of acrocentric chromosomes), ring chromosomes, and marker chromosomes
G-11 Banding protocol
Giemsa at pH 11. An alkaline treatment of the chromosomes causes loss of the Giemsa binding sites
G-11 Banding stains
pericentromeric regions of all chromosomes, the heterochromatin regions of chromosomes 1, 9, 16, and the distal Yq, and acrocentric chromosome satellite regions
G-11 Banding is used to
differentiate between human (pale blue) and rodent (magenta) chromosomes in hybrid cells
DAPI is
4,6- Diamidino-2-phenylindole. a fluorescent dye
Where does DAPI bind
DNA minor groove with an affinity for A + T-specific binding.
What is Distamycin A
Distamycin A is a DNA-binding, oligopeptide antibiotic
What does Distamycin A bind to
A + T-specific DNA but is non-fluorescent
DAPI-Distamycin A staining principle
two dyes have similar base-pair binding preferences but non-identical binding affinities and dissimilar structures “competitive binding”.
DAPI-Distamycin A staining advantages
Only staining technique that highlights 15p, quick protocol.
DAPI-Distamycin A staining disadvantages
Fades quickly therefore no permanent preparation
Counterstaining used to
enhance banding patterns that do not have a very high resolution
Counterstaining mechanisms
oElectron energy transfer from the primary stain to the counterstain. must be spectral overlap of the fluorescence emission of the primary stain and the acceptor absorption range of the counterstain. As a result the fluorescence from the first fluorochrome is quenched.
o Direct binding competition: involves the selective displacement of the primary stain by the counterstain. e.g. Distamycin binds to all the chromosomes except those regions stained with DAPI
what is BrdU
5’-Bromo-2’deoxyuridine- a thymidine analogue that is readily incorporated into chromosomes
Replication banding / Sister Chromatin Exchange (SCE) uses
BrdU
