Detecting Chromosomal Abnormalities Flashcards
What are ways of sourcing a prenatal sample for testing?
Amniocentesis - sample of amniotic fluid
Chorionic villus sampling - sample from placenta
Cell free fetal DNA
What are the ways of sourcing of a postnatal sample for testing?
Blood
Saliva
Describe the use of chromosome staining.
Most common = G-banding
G = Giesma
Chromatin can exist in 2 different forms - euchromatin and heterochromatin
Euchromatin = GC rich, loosely packed, genes active
Heterochromatin = AT- rich, tightly packed, genes innactive
Both chromatin stain differently
Uses a metaphase spread because chromosomes are most condensed and visible
Describe how the karyotype of a patient differs from expected.
Describe the process of analysing the karyotype of a patient using chromosome staining.
- Obtain blood sample form patient
- Add phytochemagglutinin
- Culture at 37 degrees for 3 days
- Fix cells using colchicine and hypotonic saline
- Spread cells into slide by dropping
- Stain with Giesma and digest with trypsin
- Analyse the metaphase spread
- Karyotype
Describe how FISH is used to detect chromosomal abnormalities.
Fluorescent in situ hybridisation
Hybridisation = single stranded nucleic acid binds to a new single stranded nucleic acid strand.
Uses fluorescent probes for SPECIFIC parts of the genome
Takes several days at least
Involves cell culturing and metaphase spread.
Looks for aneuploidies, translocations and large deletions
What happens in FISH?
- Fluorescent probe
- Denature probe and target DNA
- Mix probe and target DNA
- Probe binds to target
- Visualise chromosomes
What is a probe?
Single stranded DNA or RNA
20-1000 bases in length
Labelled with fluorescent or luminescent molecule
Describe how Array CGH (comparative genomic hybridisation) is used to detect structural abnormalities in chromosomes.
For detection of sub microscopic chromosomal abnormalities
Uses extracted DNA
Uses thousands of probes for different parts of the genome
Patient DNA labelled green
Control DNA labelled red
What occurs during Array CGH?
- Patient and control DNA are labeled with fluorescent dyes and applied to microarray
- Patient and control DNA compete to attach or hybridise to micro array
- The microarray scanner measures the fluorescent signals
- Computer software analysis the data and generates a plot
Describe how QF-PCR is used to detect chromosomal abnormalities.
Quantitative fluorescence polymerase chain reaction
Used to see how many copies of a chromosome a patient has.
Uses extracted DNA
Quick ~48 hours
Trisomies 13, 18, 21
Looks for aneuploidies
NEED TO KNOW WHAT YOUR LOOKING FOR
What are microsatellites?
Short repeated sequences distributed across the whole genome
Most are not within genes
Number of repeats varies between induviduals
Total length of micro satellite sequence varies between induviduals. (Di, tri or tetra nucleotide sequence with variable number of repeats)
Short tandem repeats are microsatelittes = simple sequence repeat (SSR)
Describe how we can detect microsatellites.
- Isolate DNA from induvidual
- Design primers specific to flanking sequence
- PCR amplification of micro satellite region
- Gel electrophoresis - genotype size of fragments
- Homozygous - single product of a specific size. Heterozygotes = two different sized products.
What is PCR?
Exponential amplification of a DNA fragment of known sequence.
Consists of incubating at three different temperatures
- denaturation
- annealing
- extension
What are the components for a PCR reaction?
Template – DNA to amplify
Primers – Short pieces of ssDNA (15-30bp)
Polymerase – thermostable enzyme (Taq)
Nucleotides – single base mixture (dNTPs)
Buffer – To maintain pH
MgCl2 – Essential for polymerase activity
What is PCR thermal cycling?
Thermal cycling temperature for PCR
