Techniques of Cytogenetic Analysis Flashcards
Whole genome tests
G-banding
Next generation sequencing
Microarrays
Targeted testing tests
FISH
MLPA
QF-PCR or qPCR
G-banding process
- Cell culture
- Mitotic arrest
- Hypotonic
- Fixation
- Trypsin and Leishman’s stain
- Banding AT&GC rich regions
FISH stands for
Fluorescent in situ hybridisation
MLPA stands for
Multiplex ligation dependent probe amplification
Array CGH stands for
Microarray comparative genomic hybridisation
QF-PCR stands for
Quantative fluorescent polymerase chain reaction
Process of FISH
- Label chromosomes
- Denature chromosomes
- Hybridise
- Washing
- Visualisation
- UV light
Types of probes
Unique sequence
Centromeric
Paints
Applications of FISH
copy number imbalance Aneuploidy Confirmation/clarification of G-banding Confirmation of array CGH Identifying specific abnormalities in cancer
MLPA summary
Multiplex ligation-dependent probe amplification
DNA-based
Multiplex PCR
Copy number changes up to 50 different genomic locations simultaneously
Alternative to FISH
MLPA schematic
- Probe added to test DNA and allowed to hybridise
- Ligation reaction
- PCR amplification using a single primer pair
Microarray CGH summary
Genome-wide screen
Hybridise sample and DNA to microarray chip with 1000s of oligonucleotides
Genomic imbalances (copy number variants) at high resolution
Higher detection rates
Replaces karyotyping as 1st line test
Microarray CGH process
- 3ml of blood added to control DNA of same sex
- Co-hybridise
- Add to microaway
- Put through scanner
- Assess colour ratios
- Analyse results
Advantages of array CGH
Early diagnosis - 1st line test
High resolution = higher diagnostic hit rate
Greater accuracy of location/size of imbalances
Information on relevant genes
Disadvantages of array CGH
low level mosaics not detected
non-pathogenic and uncertain pathogenic changes detected
needs good quality DNA
QF-PCR summary
PCR amplification of short tandem repeats using fluorescent primers.
Products visualised and quantified as peaks
Prenatal aneuploidy detection process
- DNA extraction from amniotic fluid/chorionic villi
- PCR aplification to chromosome 13, 18, 21, X&Y
- DNA dosage is up to 4-5markers/chromosome
Size of product is directly related to number of ATTT repeats + repeat number is highly variable
Microsatellite tetranucleotide repeat marker (STR)
Chromosome 21 maternal and paternal. Each box has a tetranucleotide repeat. Paternal has 4 and maternal has 6. Look at relation between 4 and 6 peaks to see whether chromosome repeated.
PCR of tetranucleotide repeat is analysed using what?
Fluorescent sequencer - a polyacrylamide gel to separates products by size
What is done after the tetranucleotide test is done?
Report made results of the test are found within 24hours.
Karyotype follow-up
What is qPCR?
Real time PCR Quantitative comparison vs reference gene & normal control patient Confirms small CNVs Used when FISH unsuitable Primer design
What is a RQ?
Relative Quantitation
A method of qPCR quantification which compares patient sample vs normal control which is assessed by 2 different primer sets.
What do you do to clarify imbalances identified via this testing?
Find specific size of imbalance
Trawl genomic databases
OMIM morbid genes or not - check against phenotype
FISH follow up for confirmation, parental studies
Supportive literature (leaflets)
What sort of fertility problems can people get?
Recurrent miscarriage (x3) - 2-3% carry balanced chromosome change Infertility - 10% of infertile males have chromosome change
Methods of prenatal diagnosis
Amniocentesis (16w)
Chorionic villus biopsy (CVS, 12w)
NIPT (12w)
What does CVS stand for?
Chorionic villus sampling
What does NIPT stand for? and what is it?
Non-invasive prenatal testing (NIPT)
Maternal blood sample to extract circulating free foetal DNA
Assesses aneuploidy of 13,18,21
Risk for aneuploidy = invasive test to confirm
Indicators for prenatal diagnosis
Higher maternal age
Serum screen risk
Abnormal ultrasound scan (USS)
FH/previous chromosome abnormality
Cytogenetics and amniotic fluid testing process
- Portion for DNA extraction (QF-PCR)
- Separate cells from remaining fluid
- Culture cells (7-14 days) if QF-PCR result abnormal
- G-banded analysis
Array CGH and prenatal diagnosis
Replacement of cell culture if abnormal scan
Pro: increased resolution and detection rate
Con: ethical e.g. small duplication & associated incense + needs parental follow up
What genetic change is there in leukaemia?
Novel fusion gene
abl (9th chromosome); bcr (22nd chromosome) bcr/abl fusion gene = abnormal protein.
FISH detection of translocations
Produces fusion signal e.g. abl/bcr probes
So, normal FISH looks like 4 blobs. 2 green 2 red = normal. Abnormal = 2 r/g; one red and one green.
What is the MYCN gene
A gene responsible for neuroblastoma
