Application of Mol Bio to Medicine Flashcards
(24 cards)
What are the 4 mechanisms of cytogenetics?
- G-banding
- FISH
- QF-PCR
- Array-CGH
What are the 3 most common trisomies (extra chromosome)?
- Down syndrome (21)
- Edward syndrome (18)
- Patau syndrome (13)
What are the 3 key features used to identify chromsomes?
- size
- banding pattern
- centromere position
What are the 4 different centromere localisations?
Telocentric we don’t get in humans
Why do we look at chromosomes in metaphase?
As they are the most visible and condense, and duplicated as well
What are the two different forms of chromatin?
Euchromatin + heterochromatin
Euchromatin = GC-rich, loosely packed, genes active
Heterochromatin = AT-rich (darker), tightly packed, genes inactive
What is the most common form of chromosome banding?
G-banding (G=giemsa)
Summarise G-banding
- extract lymphocytes
- culture at 37C for 3 days
- add colchicine to arrest in metaphase
- add hypotonic saline to cause cell swelling
- cells lyse
- mount on slide + stain
- look for aneuploidies, translocations + v large deletions
Discuss FISH
Do the same as G-banding, except you denature the fluorescent probe and target DNA, then mix the probe with target DNA and the probe should bind to the target
What 3 syndromes does FISH test for?
- 22q deln syndrome (chromosome 22)
- Prader-Willi syndrome (chromosome 15)
- Cri-du-chat (chromsome 5)
Often deletions - FISH allows us to see these problems with greater clarity
What is spectral karyotyping?
Essentially chromosome painting allowing to use multiple probes across the genome
Which test uses micosatellites?
QR-PCR, microsatellites are repetitive bits of DNA across genome, most do nothing but exist - helpful as markers for various things, eg. gene mapping, diagnostic tests as we know where they are.
Number of repeats is constant within one individual but varies between different individuals.
What type of abnormality is QF-PCR useful for?
Trisomies!
What are the 3 main stages of QF-PCR? (ADA)
- Amplification
- Detection
- Analysis
What are key features of QF-PCR?
- for trisomies
- uses fluorescent probes for SPECIFIC microsatellite markers on SPECIFIC chromosomes
- uses extracte dDNA
- quick (48 hrs)
- looks for enuploidies
What is Array-CGH used for?
Sub-microscopic chromsome abnormalities, for microdeletions, microduplications, CNVs, the main indication is developmental problems, dysmorphia
How can copy number variation be helpful and detrimental?
Refers to duplications and deletions
Can protect. eg from HIV
Can be detrimental eg. autism + schizophrenia
Many often do nothing
Describe the process of aCGH
- extract DNA
- test DNA labelled w/ fluorescent dye
- control DNA lebelled w/ diff fluo dye
- mix DNA together
- apply denatured DNA to array -> hybridise
- measure relative fluorescence
What are the separate steps of Sanger Sequencing?
- amplify (PCR)
- denature/strand separation
- anneal primer
- extension
- termination
- denature
- read
What components are needed for Sanger sequencing?
- dideoxy or chain termination method
- dna template
- dna primer
- dna polymerase
- dNTPs
- ddNTPs (lacks OH)
How does a ddNTP result in termination during sequencing?
ddNTPs lack OH group, which is important for formation of the nucleotide backbone, so therefore result in termination of the strand
Breaking it down further, what are the 4 steps of the sequencing part of the reaction?
- strand separation
- anneal primer
- extension
- termination
How is Next Generation Sequencing different from Sanger sequencing?
It’s targeted, looks at whole exome and genome - identifies substitutions as well and can look at ALL genes at once.
Summaries the differences in between the 6 tests we’ve looked at
