What is Cystic Fibrosis?
A genetically inherited disease which involves the build up of thick, sticky mucous
Which body system(s) being affected by CF?
What causes cystic fibrosis?
Which gene is affected?
How many variants have been reported?
Which variant is the most common one for cystic fibrosis?
Why can cystic fibrosis cause mild to severe damage to the body system?
How to diagnose cystic fibrosis?
Which method is used to genetically screen for cystic fibrosis?
Amplification Refractory Mutation System (ARMS)
What is ARMS? What is it based on?
Can detect single oligonucleotide changes. Based on allele-specific oligonucleotide primer design to discriminate between normal and mutant alleles.
What primers do you need for ARMS?
One gene-specific
One mutant-specific
Does ARMS require a known mutation?
Yes
Why do we need 2 primers?
Allele-specific primer (mutant specific) will only base pair with the mutant form and not the normal form or vice versa.
What type of DNA polymerase is required for ARMS?
It must lack proofreading activity e.g. TaqI polymerase
During ARMS data analysis, what does the green and blue labels indicate?
Green label - Normal Sequence
Blue label - Mutant Sequence
What does a single green peak indicate?
Homozygote wild type
What does a single blue peak indicate?
Homozygote Mutant
What does a green AND blue peak indicate?
Heterozygote
What does multiplex PCR simultaneously test for?
Several known CF mutations
What does each region of the gene produce? How can they be analysed?
Distinct size fragment.
Capillary Gel Electrophoresis (electropherogram)
What is Oligonucleotide Ligation Assay used for?
To identify point mutation
What does the OLA rely on?
DNA ligation
What does OLA consist of?
DNA/ RNA template for hybridization
Fluorescently labelled DNA probe (common probe, mutant-specific)
Allele-specific oligonucleotide (ASO, usually 15–21 nucleotides base in length)
Mobility modifier (for multiplex detection)
Ligase
How does OLA detect the mutation?
Capillary Gel Electrophoresis
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CC DNA Cloning1
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CC Synthetic Biology0
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CC Quick Recap of Terminology19
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CC Simple Mendelian Inheritance30
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CC Genetic Testing28
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CC Y Chromosome Deletions and Infertility18
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CC Quantitative Fluorescent PCR (QF-PCR)36
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CC Multiplex ligation-dependent probe amplification (MLPA)27
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CC Triplet repeat-primed PCR (TP-PCR)21
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CC All Molecular Diagnostic Methods29
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CC Sources of DNA Damage31
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CC Single Nucleotide Changes48
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CC Basis of Genetic Variation90
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Amplification Refractory Mutation System ARMS34
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Oligonucleotide Ligation Assay OLA14
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Sequence Tagged Sites (STS-based) PCR23
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Multiplex Ligation-Dependent Probe Amplification MLPA20
