20.02.02 ARMs, OLA, Pyrosequencing Flashcards
1
Q
What is known mutation detection
A
Methods that can distinguish two alleles based on a single nucleotide difference
2
Q
Uses of known mutation detection
A
- Diagnosis of diseases with limited alleleic heterogeneity
- Detection of common pathogenic variants
- Diagnosis in a family, where a mutation has been characterised
- SNP genotyping
- Testing normal controls to see if a candidate variant is pathogenic or a polypmorphism
3
Q
What is allele-specific PCR
A
- Method that allows efficient discrimination of SNPs.
- Common reverse primer and two forward allele-specific primers. Creates two allele-specific PCR products of different lengths, which are separated by electrophoresis.
- If the 3’ end nucleotide is not perfectly base paired then amplification will not take place.
4
Q
What is ARMS
A
- Amplification refractory mutation system
- An allele-specific PCR
- Paired PCR reaction, where there is a common primer with either a normal or mutant-specific primer.
- Allele-specific primers differ in their 3’ end nucleotide. Amplification does not occur if 3’ end nucleotide doesn’t match.
- Primers can be designed to give products of varying sizes so they can be multiplexed and separated by electrophoresis.
5
Q
An example of an ARMS test
A
- CF-EU Elucigene kit.
- Tests for the common 50 pathogenic variants in CFTR gene.
- Two parallel PCR reactions run in parallel
6
Q
Advantages of ARMS
A
- Quick, sensitive, simple, cheap
- Doesn’t require specialist equipment
- Can detect point mutations, insertion or deletions
7
Q
Disadvantages of ARMS
A
- Cannot detect rare or novel variants
- Polymorphisms under primer site can affecting primer binding.
- Designing assay can be complex, commercial kits are often required.
- Cross reactivity may pick up the wrong pathogenic variant. e.g. Phe508del and Phe508Cys
- Prone to contamination (e.g. MCC in prenatals)
8
Q
What is restriction enzyme digest
A
- Where restriction enzymes make double stranded breaks in DNA at specific recognition sites.
- Used when a substitution creates or abolishes a recognition site of a restriction endonuclease.
- PCR products containing the potential variant are digested and products separated by electrophoresis to see if a cut has occurred.
- Methylation specific enzymes can be used to digest methylated DNA.
9
Q
Example of restriction digests in diagnostic labs
A
- Haemochromatosis. C282Y variant using Rsa I.
- Haemophilia A. F8 intron 22 inversion using bcl I
10
Q
Ada vantages of restriction enzyme digest
A
- Cheap, easy, quick
- No specialist equipment needed
11
Q
Disadvantages of restriction enzyme digest
A
- Polymorphisms can affect restriction target sequence.
- Can only be used in a diagnostic lab if the variant creates a restriction site.
- Only suitable for use if a restriction enzyme is available for the sequence of interest.
- Rare or obscure restriction enzymes would not be suitable as expensive and poor quality.
- Partial or over digestion will affect interpretation.
- Non-specific activity can occur in the presence of contaminants (e.g. glycerol)
12
Q
What is FRET
A
- Fluorescent resonance energy transfer
- Uses minor groove binders (long molecules that bind to duplex with DNA at minor groove). Very stable interaction.
- Used in real time PCR.
- Polymerisation of a new DNA strand is initiated from primers. Once the polymerase reaches probe, 5’-3’ exonuclease degrades the probe. The fluorescent reporter moves away from quencher leading to an increase in fluorescence.
13
Q
Uses of FRET assays
A
JAK2 V617F mutation in myeloid disease.
14
Q
Advantages of FRET assays
A
- Highly selective to target.
- Very stable
- Can detect low levels of mutant DNA
15
Q
Disadvantages of FRET assays
A
- No multiplexing
- Costly, probes for multiple targets
