19.01.03 relationship between chromosome structure and banding

Question 1

g banding dye

Answer 1

Giemsa

Question 2

Romanowsky-Giemsa effect

Answer 2

purple coloration of chromatin DNA, contrasting with the blue-stained RNA-containing cytoplasm and nucleoli

Question 3

G-banding staining regions

Answer 3

heterochromatin, gene- poor, AT- Rich, low histone acetylation level, long Intermediate Nuclear Elements

Question 4

G-banding method

Answer 4

aged metaphase chromosome preparations treated with a protease (trypsin) or with hot 2x SSC and then stained with Giemsa stain or similar chromatin stain

Question 5

A chromosome band

Answer 5

is part of a chromosome that can be distinguished from adjacent segments by appearing lighter or darker by one or more techniques

Question 6

maximum resolution of G-banding is

Answer 6

~3-5Mb

Question 7

Differential banding pattern is due to

Answer 7

distribution of chromatin proteins to DNA , chromatin compaction and DNA strandedeness

Question 8

Reverse Banding (R-banding) stains the

Answer 8

GC-rich euchromatin

Question 9

Advantage of R-banding compared to G-banding

Answer 9

telomeric regions are generally stained more darkly, allowing for improved visualisation if telomeres are involved in aberrations

Question 10

protocol for R-banding

Answer 10

incubating chromosomes in a hot (85-90°C) phosphate buffer followed by Giemsa staining.

Question 11

Constitutive heterochromatin banding (C-banding) stains

Answer 11

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

Question 12

Constitutive heterochromatin is

Answer 12

a permanent transcriptionally-repressive state, highly polymorphic. Can affect the size and the localisation of heterochromatin without observable phenotypic effect.

Question 13

facultative heterochromatin is

Answer 13

condensed only semi-permanently via epigenetic changes which are reversible, allowing DNA transcription.

Question 14

C-banding uses

Answer 14

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.

Question 15

C-banding protocol

Answer 15

involves alkali treatment (usually Barium hydroxide as milder than NaOH) for DNA denaturation

Question 16

Q- Banding uses

Answer 16

Quinacrine dihydrochloride fluorochrome.

Question 17

Quinacrine dihydrochloride binds to

Answer 17

Adjacent A-T pairs, so A-T rich regions fluoresce brightly

Question 18

Q-banding is useful for

Answer 18

confirming the presence of Y chromosome material (distal long arm of the Y fluoresce brightly)

Question 19

Q-banding disadvantage

Answer 19

staining is prone to fading with time

Question 20

T-Banding

Answer 20

Thermal treatment is used to produce staining of strictly telomeric R-bands

Question 21

T-Banding is useful for

Answer 21

analyzing deletions or translocations involving the terminal ends of chromosomes.

Question 22

CT banding

Answer 22

heat treatment at high pH using Barium hydroxide

Question 23

Cd (Centromeric Dot or Kinetochore) Staining

Answer 23

produces a pair of dots at each centromere, one on each active or functional chromatid.

Question 24

Difference between Cd and C banding

Answer 24

C-banding will stain inactive and active centromeric regions, Cd banding will only stain active

Question 25

Cd banding is useful to

Answer 25

differentiate functional from nonfunctional centromeres and study Robertsonian translocations (centromere to centromere translocations of acrocentric chromosomes), ring chromosomes, and marker chromosomes

Question 26

G-11 Banding protocol

Answer 26

Giemsa at pH 11. An alkaline treatment of the chromosomes causes loss of the Giemsa binding sites

Question 27

G-11 Banding stains

Answer 27

pericentromeric regions of all chromosomes, the heterochromatin regions of chromosomes 1, 9, 16, and the distal Yq, and acrocentric chromosome satellite regions

Question 28

G-11 Banding is used to

Answer 28

differentiate between human (pale blue) and rodent (magenta) chromosomes in hybrid cells

Question 29

DAPI is

Answer 29

4,6- Diamidino-2-phenylindole. a fluorescent dye

Question 30

Where does DAPI bind

Answer 30

DNA minor groove with an affinity for A + T-specific binding.

Question 31

What is Distamycin A

Answer 31

Distamycin A is a DNA-binding, oligopeptide antibiotic

Question 32

What does Distamycin A bind to

Answer 32

A + T-specific DNA but is non-fluorescent

Question 33

DAPI-Distamycin A staining principle

Answer 33

two dyes have similar base-pair binding preferences but non-identical binding affinities and dissimilar structures “competitive binding”.

Question 34

DAPI-Distamycin A staining advantages

Answer 34

Only staining technique that highlights 15p, quick protocol.

Question 35

DAPI-Distamycin A staining disadvantages

Answer 35

Fades quickly therefore no permanent preparation

Question 36

Counterstaining used to

Answer 36

enhance banding patterns that do not have a very high resolution

Question 37

Counterstaining mechanisms

Answer 37

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

Question 38

what is BrdU

Answer 38

5’-Bromo-2’deoxyuridine- a thymidine analogue that is readily incorporated into chromosomes

Question 39

Replication banding / Sister Chromatin Exchange (SCE) uses

Answer 39

BrdU