FISH

Question 1

denaturation methods

Answer 1

heat and formamide/salt

Question 2

increasing the formamide concentration ___ the temperature DNA denatures

Answer 2

decreases

Question 3

Conditions that favor DNA denaturation are known as…

Answer 3

stringent

Question 4

What is the purpose of the salt when exposing DNA to heat and formamide?

Answer 4

stabilize the single-stranded DNA (monovalent cations)

Question 5

4 parameters that influence DNA Tm and reannealing

Answer 5

temp, pH, [monovalent cations], formamide

Question 6

What is the purpose of the ethanol series after denaturation?

Answer 6

Prevents reannealing, removes residual formamide, preserves DNA morphology

Question 7

Purpose of dextran sulphate in hybridization buffer

Answer 7

effectively increases probe concentration in solution

Question 8

Purpose of herring/salmon sperm DNA in the hybridization buffer

Answer 8

blocks non-specific binding sites

Question 9

What conditions increase the stringency of post -hybridization wash?

Answer 9

high temp, longer duration, lower salt concentration

Question 10

The first post-hyb wash removes unbound probe and mismatch bound probe, the second post-hyb wash…

Answer 10

helps stabilize the probe/target duplexes

Question 11

is the stringency of the first or second post-hyb wash higher?

Answer 11

first

Question 12

Purpose of detergent (such as NP-40) in the post-hyb wash?

Answer 12

remove unbound probe

Question 13

the reporter molecule bound to the dUTPs in the probe is…

Answer 13

a fluorochrome

Question 14

dUTPs bound with fluorochromes are introduced into the probe sequence by …

Answer 14

nick translation, random-primer extension, or PCR

Question 15

Most common anomalies associated with CLL are

Answer 15

+12, del(13q), del(17p) TP53, del(11q) ATM

Question 16

Hyperchromasia

Answer 16

In H&E staining cancer cells appear darker blue/purple bc of increased division, transcription, growth signalling

Question 17

FFPE pre-treatment steps

Answer 17

1. bake
2. Xylene
3. HCl
   4: Sodium thiocyanate (NaSCN)
4. Pepsin
5. Ethanol

Question 18

Purpose of Xylene in FFPE pre-treatment

Answer 18

remove paraffin wax

Question 19

purpose of HCl in FFPE pre-treatment

Answer 19

digest extra-cellular proteins

Question 20

Purpose of NaSCN in FFPE pre-treatment

Answer 20

break protein-protein and protein-DNA cross-links

Question 21

Purpose of pepsin in FFPE pre-treatment

Answer 21

digest collagen matrix, proteins in the cytoplasm and nuclear membrane

Question 22

IHC staining: reflex for HER2 FISH when?

Answer 22

2+ (0, 1+ are negative, 3+ is positive)

Question 23

describe a normal result (RRGG) for a dual fusion BCR/ABL FISH in ISCN if 100 cells were scored

Answer 23

nuc ish(ABL1,BCR)x2 [100]

Question 24

ISCN for dual fusion BCR ABL FISH with FFRG pattern if 200 cells scored and 100 had that signal pattern

Answer 24

nuc ish(ABL1, BCR)x3(ABL1 con BCRx2) [100/200]

Question 25

Break apart probe: describe ISCN for normal pattern (FF) for MYC probe

Answer 25

nuc ish(5’MYC,3’MYC)x2(5’MYC con 3’MYCx2) [200]

Question 26

describe ISCN for MYC translocation (break-apart probe)

Answer 26

nuc ish(5’MYC,3’MYC)x2(5’MYCsep3’MYCx1) [200]

Question 27

Smallest FISH probes

Answer 27

plasmids and cosmids (~40kb)

Question 28

Medium-sized FISH probes

Answer 28

PACs and BACs

Question 29

Largest FISH probes

Answer 29

YACs

Question 30

Probe type often used for identifying aneuploidies, marker chromosomes, interphase sexing. Targets the chromosome of interest

Answer 30

Repetitive sequence probes

Question 31

Probe type that hybridizes to a locus-specific site in the genome. Used for microdeletion/duplication syndromes, rearrangements in cancer

Answer 31

Unique sequence probe

Question 32

Multiple Myeloma FISH panel

Answer 32

TP53: del(17p)
FGFR3;IGH : t(4;14)
IGH;MAF : t(14;16)