FISH Flashcards
(32 cards)
denaturation methods
heat and formamide/salt
increasing the formamide concentration ___ the temperature DNA denatures
decreases
Conditions that favor DNA denaturation are known as…
stringent
What is the purpose of the salt when exposing DNA to heat and formamide?
stabilize the single-stranded DNA (monovalent cations)
4 parameters that influence DNA Tm and reannealing
temp, pH, [monovalent cations], formamide
What is the purpose of the ethanol series after denaturation?
Prevents reannealing, removes residual formamide, preserves DNA morphology
Purpose of dextran sulphate in hybridization buffer
effectively increases probe concentration in solution
Purpose of herring/salmon sperm DNA in the hybridization buffer
blocks non-specific binding sites
What conditions increase the stringency of post -hybridization wash?
high temp, longer duration, lower salt concentration
The first post-hyb wash removes unbound probe and mismatch bound probe, the second post-hyb wash…
helps stabilize the probe/target duplexes
is the stringency of the first or second post-hyb wash higher?
first
Purpose of detergent (such as NP-40) in the post-hyb wash?
remove unbound probe
the reporter molecule bound to the dUTPs in the probe is…
a fluorochrome
dUTPs bound with fluorochromes are introduced into the probe sequence by …
nick translation, random-primer extension, or PCR
Most common anomalies associated with CLL are
+12, del(13q), del(17p) TP53, del(11q) ATM
Hyperchromasia
In H&E staining cancer cells appear darker blue/purple bc of increased division, transcription, growth signalling
FFPE pre-treatment steps
- bake
- Xylene
- HCl
4: Sodium thiocyanate (NaSCN) - Pepsin
- Ethanol
Purpose of Xylene in FFPE pre-treatment
remove paraffin wax
purpose of HCl in FFPE pre-treatment
digest extra-cellular proteins
Purpose of NaSCN in FFPE pre-treatment
break protein-protein and protein-DNA cross-links
Purpose of pepsin in FFPE pre-treatment
digest collagen matrix, proteins in the cytoplasm and nuclear membrane
IHC staining: reflex for HER2 FISH when?
2+ (0, 1+ are negative, 3+ is positive)
describe a normal result (RRGG) for a dual fusion BCR/ABL FISH in ISCN if 100 cells were scored
nuc ish(ABL1,BCR)x2 [100]
ISCN for dual fusion BCR ABL FISH with FFRG pattern if 200 cells scored and 100 had that signal pattern
nuc ish(ABL1, BCR)x3(ABL1 con BCRx2) [100/200]
