Microarrays + PCR Flashcards
What are constitutional genetic disorders?
Genetic abnormality is present in all cells from birth.
What is a Somatic disorder?
Genetic abnormality is acquired sometime after birth
Outline the basic process of FISH
- ssDNA probe complimentary to target sequence, labelled with fluorescent protein (fluorochrome)
- Hybridise 37’C, 16 hours
- Denature to allow probe access to DNA target (~70’C)
- Wash, to remove background signal, and non-specific binding
- Apply DAPI counterstain to visualise chromosomes
- Analyse with fluorescent microscope
Applications of FISH
- WHole chromosone paint
- Trisomy 21 detection
- Locus specific
- Amplification
- Fusion
Weakness of FISH
Very locus specific
What are you looking for in Microarrays
Copy number variants
Gains and losses
Duplications and deletions
Monsomy and trisomys.
Can identify which genes are invovled
What are microarrays?
DNA probes attached on an array
Then hybridise patient DNA to array (and control DNA).
Detect using fluorescence
What can microarrays not detect?
Balanced changes such as translocations
Who are tested using microarrays?
Children with develpmental delay
Prenatal analysis
Cancer
Children with developmental delay
Autism
Intellectual disability
Dysmorphic features
Congenital abnormalities
Prenatal analysis
DNA from amniotic fluid or CVS
Abnormal ultrasound scans
Microarrays have also replace karyotyping in prenatal analysis.
Cancer
Myeloid dysplastic syndrome - because the causative genetic abnormalities are most often copy number changes.
NB gene fusions visible via balanced translocations
Compare the sensitivity of karyotypes and arrays
G-band detects changes of 5-10Mb at best
Arrays improves the resolution for detection of cytogenetic abnormalities
Arrays - there are different types but resolution can be as good as 100kb
Find causative copy number, gain or loss, change in ~20% of developmental delay cases
Vs karyotype ~5% causative change found
What are the two types of arrays?
Oligonucleotide arrays
Single nucleotide polymorphism (SNP) arrays
Oligonucleotides arrays
– uses oligonucleotide DNA probes complimentary to specific regions of genome
SNP arrays
– 100,000s of SNP probes across the genome
What is compared in oligonucleotide arrays?
In oligonucleotide arrays the quantity of patient DNA bound to the arrays is compared to amount of control DNA.
What is the DNA labelled with in Oligonucleotide arrays?
Patient and control DNA is labelled with different fluorescence.
What is the process behind oligonucleotide arrays?
What type of hybridisation is this?
The DNA’s are fragmented to short DNA sequences and together they are hybridised to the oligonucleotide array.
This is a competitive hybridisation.
What does the relative intensity of fluoresence meansure in a oligonucleotide array?
The amount of patient DNA versus control DNA bound to an oligo nucleotide array is measured by the relative intensity of fluorescence.
What does it mean if the patients DNA quantity is the same as the control DNA at a given oligonucleotide? i.e (1:1)
If the patient DNA is present in the same quantity as the control DNA at a given oligonucleotide, the relative fluorescence will be the same, as in a 1 to 1 ratio.
This would indicate that at the location the oligo probe is specific for the patient is normal disomic.
What does it indicate if there is more of the patients DNA compared to control DNA at a given oligonucleotide?i.e (1:2)
If there is more patient DNA relative to the control DNA, as in the fluorescence for the patient DNA is stronger by half again, and thus a ratio of 1 to 2, this would indicate that at the location the oligo probe is specific for the patient has a gain of genetic material.
What does it indicate if there is less of the patients DNA compared to control DNA at a given oligonucleotide? i.e (2:1)
If there is more less patient DNA relative to the control DNA, as in the fluorescence for the patient DNA half that of the control, and thus a ratio of 2 to 1, this would indicate that at the location the oligo probe is specific for the patient has a loss of genetic material.
What does/does not oligonucleotide array detect?
An oligonucleotide array detects copy number variants
It will not detect genetically balanced changes, such as chromosomal translocations and inversions.
SO what to Oligonucleotide arrays detect?
detets gain or loss of patient DNA compared to control
What is a SNP
These SNPs are not mutations but normal variantation within the genome, they do not have an adverse effect on resulting protein or the individual carrier.
Points in the DNA sequence which differ at a single base
There are approximately 10 million SNPs in the human genome.
Describe SNP array
- SNP Array contains DNA fragments (probes) with known SNPs
- A SNP array consists of probes, short DNA fragments of about 25 nucleotides long, attached to the surface of the array chip.
There are two version of every probe, each with a different nucleotide, so there is a probe for the A allele and the B allele for a given SNP. - Each allele, A+B, represented on array
- Patient may be AA, BB or AB
What do SNP arrays not have?
Control DNA
How do SNP arrays work?
SNP arrays work by labelling one allele green and the alternative allele red
The colour of the fluorescence is detected at each probe, and this indicates which nucleotide is present.
For example, If the patient DNA binds to the probe it releases the fluorescence, detected by the scanner, a red signal may indicate a T allele, whereas a green signal may indicate a C allele.
What indicates heterozygosity for the SNP?
both probes emit fluorescence
What indicates homozygosity for the SNP?
Only one probe emits fluorescence
Outline basic process of SNP array
Amplify & digest/fragment patient DNA
Hybridise to array
Image scanning
Identify which SNP fluoresces - The fluorescence at each probe site is recorded and the relative fluoresence of A vs B is plotted in the order they occur along a chromosome.
Gains And losses can be detected.
A, B or both
What do/do not SNP array detect?
SNP arrays can detect loss of heterozygosity. This refers to large tracts of the genome where there are no heterozygous SNPs. This can be as large as a whole chromosome.
Balanced chromosome abnormalitesd cannot be detected
What does a loss of hterozygosity show?
As loss of heterozygosity shows that there is less normal variantion within that individuals genotype.
Why is loss of heterozygosity important?
Loss of heterozygosity is important as it can unmask recessive gene mutations that may cause disease. For example, if the parents are related and each carry a familial recessive gene variant in one allele, any offspring wouild be at risk of inheriting the same allele from each parent (same chromosome) and therefore being affected with the disorder.
What happens if there is a deletion - SNP array
If there is a deletion, each SNP will show only an A allele or only a B allele.
there will be no heterozygous SNPs – no AB.
What happens if there is a duplication? (SNP array)
If there is a duplication, each SNP will show AAA, BBB, AAB or ABB: BUT- no AB
What are the gains and losses identified from microarrays called?
These are referred to as copy number variants, or CNVs,
What is a copy number variant?
is a normal occurrence in the human genome, this is normal copy number variation.
What is the general rule in genetics about loss and gain?
A general rule in genetics is that a loss is more likely to have a phenotypic consequence than a gain.
Do gene(s) fit with referral reason
A good question to consider is whether the gene or genes affected by the CNV can be linked to the referral reason.
Do parents have same change? (CNV)
Testing whether parents have the same CNV can be highly informative.
If the parent has the CNV and also has similar or the same clinical evidence then this is good supporting evidence that the CNV is causative.
Or if the parent has the CNV but does not share any clinical symptoms with the child, this provides evidence that the CNV is less likely to be causative,
Is it seen in normal population? (CNV)
As for DNA variant analysis, a strong indicator of whether a CNV is likely to be causing disease is whether it is seen in the normal population
Why must we consider penetrance? What is it? (CNV)
But, we must consider penetrance, which is a measure of the likelihood of a given genetic variant causing a disease.
The reality is that a DNA variant or a CNV does not always cause outward medical problems, sometimes a child may display symptoms whilst the parent who has the same genetic variant has no symptoms.
Perspective: Karyotype and FISH
Karyotype + FISH
large abnormalities >3-5Mb
Balanced changes
10% mosaicism detection
~5% of patients diagnosed
Perspective: Arrays
Arrays
Smaller abnormalities <200Kb
Cannot detect balanced changes
10-30% mosaic detection
10-15% of patients diagnosed
Genome sequencing
Single nucleotide DNA variants
Single nucleotide DNA variants
With bioinformatics can do copy number and balanced changes!
Mosaicism dependent on depth of coverage (number of times a DNA fragment is seqenced
Up to 30% patients diagnosed
New genes
Lots of data!
Further reading
Deciphering Developmental Disorders https://www.ddduk.org/
Understanding Rare Chromosome and Gene Disorders https://www.rarechromo.org/
NHS Genomics Education Programme https://www.genomicseducation.hee.nhs.uk/
Association of Clinical Genetic Science best practice guidelines https://www.acgs.uk.com/quality/best-practice-guidelines/
What is the starting point to many of the assays?
Polymerase chain reaction (PCR)
What does PCR do?
PCR is used to amplify regions of the genome we are particularly interested in.
What are the target section of PCR?
Target regions can be a
- single gene
- part of a gene or
- non-coding sequence
The DNA targeted maybe parts of a single gene, including exons or noncoding regions.
What can the amplified DNA be used for?
This DNA can then be used for further testing such as sanger sequencing or genotyping for the size of the fragments.
What does PCR use
Fluorescently labelled primers
PCR process
Denaturation – the double-stranded DNA template is heated to separate it into two single strands
Annealing – the temperature is lowered to enable primers to bind to the template DNA
Elongation/extension – the temperature is raised to promote the Taq polymerase enzyme to produce the new DNA strand
What are the two types of PCR used in genetic diagnostics?
quantitative fluorescent PCR, or QF-PCR/
What is Quantitative Fluorescent (QF)-PCR used for?
For aneuploidy (gains, loss of chromosome)
Prenatal 24hour test for Down syndrome
DNA from amniotic fluid
Describe QF-PCR
Amplify and quantify
Uses Fluorescently labelled primers
Targeting small tandem repeats (STRs)
STR = repeats of two or more nucleotides e.g. 10 repeats of GAC
The number of repeats varies between individuals(alleles)
e.g. STR on chromosome 21
Allele 1 (maternal chr) = 5 repeats
Allele 2 (paternal chr) = 9 repeats
How does the QF-PCR prcoess amplify both alleles
Relative copy number of each allele = the ratio of the peak areas or peak heights detected for each STR
i.e. how much of allele 1 versus allele 2
Normal / Disomy = 2 alleles (peaks) in 1:1 ratio
Trisomy = 3 alleles in 1:1:1 ratio or 2 alleles in a 2:1 ratio (i.e. 2x copy number of 1 allele vs the other allele)
What is used for mutation testing?
Digital droplet PCR
What is digital droplet PCR?
Detecting a specific mutation
Partition a DNA sample into many individual real-time PCR reactions
A portion of the reactions contain the target sequence of interest
Allows for the detection of low level mutations in a background of wild-type
Digital droplet PCR process
Probe binds, as PCR performed fluoresence separated from quencher & released
Droplets are ‘counted’ based on the fluorescence within the droplet
A droplet can either be positive (contains the target mutant sequence) or negative (wild-type sequence)
Describe ddPCR analysis
The ddPCR assay will typically contain four clusters of droplets (assuming that the mutation is present)
Double negative droplets: contain no targeted DNA templates
Wild-type only droplets
Mutant only droplets
Double positive droplets: contain both wild-type and mutant DNA templates
Digital droplet PCR video
Digital droplet PCR https://www.youtube.com/watch?v=WU3qKhIUc54
