L10&11- Techniques of chromosomal analysis Flashcards
Whole genome test types
G-banding
Next ten sequencing
Microarrays
Targeted testing
FISH
MLPA
QF-PCR/qPCR
Process of G banding
Cell culture –> Mitotic arrest –> Hypotonic –>Fixation –>Trypsin & Leishman’s stain –>Banding - AT and GC rich regions
Molecular cytogenetic techniques
Fluorescent in situ hybridisation (FISH)
Multiplex ligation dependent probe amplification (MLPA)
Microarray comparative genomic hybridisation (array CGH)
Next generation sequencing
Quantitative fluorescent PCR (QF-PCR)
qPCR
Fluorescent in situ hybridisation (FISH)
Detection of DNA material on slides using fluorescent
Dyes & UV light
FISH process
Labelling (fluorochrome) the probe (which will bind to the target you are looking for e.g chromosome 18 or X/Y) –> denaturation –> mix in with the target (sample) —> hybridisation –> (post hybridisation washing) –> Visualisation (24 hours) with UV light
Types of FISH probes
- unique sequence (specific place on an arm?)
- Centromeric (middle of the chromosome will light up)
- Paints (whole chromosome lights/painted up!)
Applications of FISH
Copy number imbalance
Aneuploidy
Confirmation/ clarification of G-banding
Confirmation of array CGH
Identifying specific abnormalities in cancer
Copy number variability
A copy number variation (CNV) is when the number of copies of a particular gene varies
For example, the chromosome that normally has sections in order as A-B-C-D might instead have sections A-B-C-C-D (a duplication of “C”) or A-B-D (a deletion of “C”)
One gene or contiguous genes
Pathogenic or benign
Molecular cytogenetic methods to assess copy number
FISH
MLPA
Microarray CGH
Next generation sequencing
QF-PCR
qPCR
MLPA
Multiplex Ligation-dependent Probe Amplification
DNA-based
Multiplex PCR
Copy no. changes in up to 50 different genomic locations simultaneously (allows multiple targets to be amplified)
Alternative to FISH
MLPA process
You have two primers (X and Y one of which has a stuffer sequence) –> added to the DNA sample and allowed to hybridise –> ligation reaction occurs –> the two parts of the primers (two parts of hybridised probe) are ligated by a thermostable ligase –> PCR amplification using a single primer pair
(More in detail regarding probes) The probes are hybridized against the target DNA and subsequently ligated. Only if ligation happened, a functional PCR strand appears, so that amplification only happens if target DNA is present in the sample. The amount of PCR product is proportional to the amount of target DNA present in the sample, making the technique suitable for quantitative measurements
MicroArray CGH (comparative genomic hybridisation)
Genome-wide screen
Hybridise sample & control DNA to a microarray “chip” 1000s of DNA spots (oligonucleotides)
Genomic imbalances (copy number variants) at high resolution (10-10000x conventional cytogenetics)
Increased detection rates
Replacing karyotyping as 1st line test
What are the requirements for MicroArray CGH?
3ml blood in EDTA (also 1-2ml lithium heparin blood for cell culture if follow up studies needed)
Control DNA from same sex
MicroArray CGH measures?
Copy number variability…..mostly imbalances
At least 3 oligonucleotides required for any call
Call imbalances >150000 bases, ie 150 Kb
Database searches to ascertain pathogenicity of imbalances
Advantages of array CGH
Early diagnosis -1st line test, reduces need for other tests and avoids the “diagnostic odyssey”
High resolution = increased diagnostic hit rate
Greater accuracy of location/size of imbalances
Information on relevant genes
Disadvantages of array CGH
Dosage changes only (Gene dosage is the number of copies of a particular gene present in a genome) – not balanced rearrangements or mutations (usually only detects unbalanced chromosomal abnormalities)
Low level mosaics not detected
Non-pathogenic & uncertain pathogenic changes detected
Needs good quality DNA
What do Next gen sequencing data analysis mean
Increased test:control ratio = gain
Decreased test:control ratio = deletion
Quantitative fluorescent PCR (QF-PCR)
PCR amplification of short tandem repeats (STRs e.g Microsatellite tetranucleotide repeat marker) [chromosome-specific, repeated DNA sequences] using fluorescent primers
Products visualised & quantified as peak areas using an automated DNA sequencer
PCR of tetranucleotide repeat analysed on a fluorescent sequencer
Prenatal aneuploidy detection
Quantitative fluorescent PCR (QF-PCR)
DNA extraction from amniotic fluid or chorionc villi
PCR amplification – primers from chromosomes 13, 18, 21, X and Y
DNA dosage in up to 4-5 markers/chromosome
aneuploidy =>2 markers with abnormal dosage
qPCR (real time PCR)
Quantitative comparison vs reference gene & normal control patient (amplify & quantify)
Confirming small CNVs
When FISH unsuitable
Primer design
qPCR quantification
Relative Quantitation (RQ) - compares difference in concentration between patient sample & normal control assessed by 2 different primer sets
RQ value is expressed as a ratio relative to 1 - a deletion has an expected value of 0.5 & a duplication an expected value of 1.5
What would qPCR show for trisomy 21?
Trisomic 2:1 ratio
Robertsonian translocation
rare form of chromosomal rearrangement that, in humans, occurs in the five acrocentric chromosome pairs, namely 13, 14, 15, 21, and 22
Look more on robertsonian translocation in lectures 10&11
slides 50 and on
Robertsonian chain trivalent
formation of a Robertsonian translocation involving chromosomes 14 and 21, and the formation of a trivalent in meiosis
Prenatal diagnosis procedures
- Amniocentesis (16w) –> QF PCR –> G banding
- Chorionic villus biopsy (CVS, 12w) –>QF PCR –> G banding
- NIPT (12w) -Non-Invasive Prenatal Testing
Non-invasive prenatal testing (NIPT)
Maternal blood sample
Extract circulating free fetal DNA
Assess aneuploidy of 13, 18, 21 (NGS)
Risk for aneuploidy – invasive test to confirm
Reduces no. of invasive tests
Array CGH and prenatal diagnosis
Replacement of cell culture if abnormal scan
Advantages
Increased resolution
Higher detection rate
Disadvantages
Ethical, eg small duplication & associated autism
Need parental follow up
Translocation in leukaemiaEg t(9;22) in CML (chronic myeloid leukaemia)
Philadelphia chromosome - is a specific genetic abnormality in chromosome 22 of leukemia cancer cells
This chromosome is defective and unusually short because of reciprocal translocation of genetic material between chromosome 9 and chromosome 22, and contains a fusion gene called BCR-ABL1.
