Chromosome Banding Flashcards
(10 cards)
What is fixation essential in banding
It removes some Histone (H1)/ non-Histone proteins and depurinates DNA
How does trypsin digestion work
Digestion is more severe in GC rich areas compared to AT Rich areas. The dye interacts with the DNA and forms a ppt. It preferentially interacts in hydrophobic areas which are the AT rich non digested regions.
AT areas: dark
GC area de: light
Describe G banding
Proteolytic digestion of chromosome followed by staining with DNA specific dye
What are the stages of c banding
Fixative (depurinates the DNA).
Alkali treatment: barium hydroxide (hydrolyses depurinated DNA and removes histones).
Salt: saline SSC ( extracts hydrolysed DNA and soluble proteins).
Stain: leishmans/ giesma (stains hetero chromatin)
What’s c banding used for
Only stains constitutive heterochromatin
1qh, 9qh, 16qh, Yqh.
Use to ID constitution of markers/ large heterochromatin regions.
What does NOR staining stain
Active NORs darkly (6-8/cell)
What are the stages of NOR staining
Incubate slides in 50% silver nitrate with gelatine.
Results in the reduction of silver nitrate to metallic silver by protein sulphdryl groups
Stain with giesma (stains proteins adjacent to NOR)
Describe replication staining
Add 5’-bromodeoxyuridine (BrdU) in DNA synthesis
BrdU= analogue of thymidine & so is incorporated.
Add BrdU late in s phase and its incorporated into late rep DNA (eg XCI).
DNA incorporating BrdU: light (giesma) & quenched fluorescence (hoechst)
Describe harlequin staining
Add 5’-bromodeoxyuridine (BrdU) in DNA synthesis
BrdU= analogue of thymidine & so is incorporated.
Leave BrdU for 48-72 hours so entire daughter strands are incorporated. After 2 rounds expose cells to UV and stain with giesma.
What does harlequin staining look for and what’s considered normal
Sister chromatid exchange.
Normal: 5-8 SCE
Abnormal: 10x more SCE
