hodgson Flashcards
what is the g banding protocol
- cells cultured to generate mitotic cells
- arrest cell cycle in metaphase
- swell nuclei with hypotonic solution
- kill cells using fixative
- drop fixed sample onto glass slide
- trypsin digest
- leishmans stain
- image analysis
why do bands appear dark and light
dark bands are AT rich - open chromatin
pale bands are GC rich - closed chromatin
what does fixative do
block chromosome condensation, kill everything in sample eg removes infectious disease
what can the centromere be referred to as
p10 or q10
which type of fish is not used clinically in the UK
indirect label FISH
what does trisomy 18 lead to
edwards syndrom
wha does trisomy 13 lead to
pateu syndrome
what does a small deletion of 5p lead to
cri du chat
what does banding resolution depend on
- cell cycle stage
- tissue sample
- experimental (slide aging, staining time, chromosome spread)
what are the steps to direct labelling FISH
- take microscope slide with DNA
- make target DNA single stranded by heating sample to 75-78 degrees
- Anneal probe at 37-40 degrees
- series of washes to remove unbound probes
- DAPI used as a counter stain
what are the 3 main types of FISH probe
- chromsome enumeration probes for common aneuploidies
- microdeletion probes
- whole chromosome pait
when would you use whole chromosome paint
when youre unsure of origin of a chromsome
At high concentrations what inhibits ribinucleotide reductase which converts CDP to dCDP meaning the concs of dCTP become rate limiting and the lymphocytes remain in S phase
dTTP
how can we cause S phase synchronisation
high levels of dTTP
how is the thymidine block released
washing (centrifugation)
- addition of dCTP
which is the preferred way of releasing the thymidine block
addition of CTP to bypass the need for ribonucleotide reductase (what excess dTTP is limiting)
why is addition of CTP the preferred method of overcoming the thymidine block
health and safety risk to centrifugation - can fracture a tube and a sample may be contaminated with infectious disease
what is another way of synchronising cells in S phase
addition of Fdu
how does addition of Fdu cause cells to be synchronised in S phase
excess Fdu blocks the synthesis of dTMP which is a precursor of dTTP so reduces its availability for DNA synthesis
how can the Fdu block be released
addition of excess dTTP but addition of too much can block the cycle again by inhibiting ribonucleotide reductase
what is colcemid
microtubule inhibitor that arrests cells in metaphase
how does colcemid inhibit microtubules
binds to soluble tubulin, colcemid-tubulin complex may still polarise but with significantly reduced efficiency
microtubule stability is reduced, preventing spindle formation
what are the 4 main sections of quality
accuracy, precision, specificity and sensitivity
what do we mean by accuracy
a test is accurate when the true abnormality is identified