Detecting Chromosomal abnormalities Flashcards
How do we detect genetic abnormalities in foetus/babies
Pre-natal
- Amniocentesis
- Chorionic villus sampling
- Cell-free fetal DNA
Post-natal
- Blood
- Sample
Chorionic Villus Sampling (CVS)
Prenatal test used to detect birth defects, genetic diseases, and other problems during pregnancy. During the test, a small sample of cells (chorionic villi) is taken from the placenta where it attaches to the wall of the uterus
· 11-14 weeks
· Miscarriage rate 0.5%-1%
· Risk of maternal contamination
· Transverse limb defects
Amniocentesis
Prenatal test in which amniotic fluid is removed from the uterus for testing or treatment. It involves removing and testing a small sample of foetal cells from amniotic fluid.
· >16 weeks
· biochemical diagnosis possible
miscarriage risk (0.5-1%)
What is G-banding?
G-banding (Giemsa banding) is a technique used in cytogenetics to produce a visible karyotype by staining condensed chromosomes.
How is G-banding done?
· Giemsa stain
· Metaphase spread (cultured cells)
· Line-up to identify chromosomes based on:
Ø Size
Ø Banding
Ø Centromere position
How can we detect an abnormal karyotype?
G-banding FISH QF-PCR NGS Array CGH
Why are there darker and lighter bands in G-banding?
The darker bands are heterochromatin, whereas the light bands are euchromatin. This is important to know because a specialist knows what to expect with regards to the banding pattern and can use that to identify which chromosome is which under the microscope.
- Euchromatin= GC rich; loosely packed; genes active
- Heterochromatin= AT rich; tightly packed; genes inactive
Fluorescence in situ hybridization (FISH)
A procedure using a fluorescence-labeled probe to detect specific nucleotide sequences within cultured cells on a microscopic slide (in metaphase spread).
1) Design fluorescent probe to chromosomal region of interest
2) Denature probe and target DNA (change temp or pH, so it goes from ds to ssDNA)
3) Mix probe and target DNA (hybridisation)
4) Target fluoresces and lights up
5) Can observe it under microscope
Uses of FISH
Used to detect large chromosomal abnormalities:
- extra chromosomes (e.g. trisomy 21)
- large deleted segments
- translocations
What makes FISH a versatile procedure?
Unlike most other techniques used to study chromosomes, FISH does not have to be performed on cells that are actively dividing
Why do we flood FISH with probes?
Probes will preferentially anneal to ss target DNA, rather than ss target DNA re-anneal with itself
What is a probe?
- ssDNA or RNA
- labelled with a fluorescent or luminescent molecule
- 20-1000 bases in length
Quantitative Fluorescence PCR (QF-PCR)
lab technique used to amplify specific DNA regions and quantify the amount of DNA present in those regions
-uses micro-satellites (should be two copies, one from each parent)
QF-PCR vs FISH
QF-PCR is quicker than FISH because it doesn’t use cultured cells
How does Array CGH work?
Microarray technique used to identify copy number variations (CNVs)- duplications & deletions (microdeletions and microduplications)
Patient DNA labelled green
Control DNA labelled red
If there is equal hybridisation between patient and control, the green and red cancel each other out and we get no signal.
Green signal= more patient DNA
Red signal= loss of patient DNA
Non-Invasive Prenatal Testing
Cell free foetal DNA (cffDNA): Foetal DNA fragments extracted from maternal plasma (10 weeks onwards)
- shedding of placental material into the maternal blood stream
- foetal DNA amplified and further tests carried out such as PCR and NGS
