Overview Of Genome Technologies In Genetic Diagnostics Flashcards
Why do you need to do PCR for diagnostics?
Amplify enough DNA molecules so we have sufficient material for downstream applications
What is fragment analysis used for?
Detect repeat expansions or other small size changes
What is fragment analysis?
PCR followed by capillary electrophoresis
What is an example of a repeat expansion disease?
Huntingtons
What is the mutation in huntingtons?
CAC repeat expansion in the huntingtin HTT gene
Why is the huntingtons mutation bad?
The resulting protein is toxic and accumulates in neurons causing cell death
How is huntingtons diagnosed?
Fragment analysis
What mutation causes cutaneous vasculitis?
R1042G mutation in gene C3
What does FISH stand for?
Fluorescent in situ hybridisation
What is FISH used for?
Detect large chromosomal abnormalities
What is the method for FISH?
Design fluorescent probe to chromosomal region of interest
Denature probe and target
Mix probe and target DNA
What does CGH stand for in array-CGH?
Comparative genome hybridisation
What colour is the patient DNA labelled in array-CGH?
Green
What colour is the control DNA labelled in array-CGH?
Red
In array-CGH, what does an increase in green signal indicate?
A gene in the patient not present in control
What does MLPA stand for?
Multiplex ligand dependent probe amplification
What is MLPA?
Variation of PCR that permits amplification of multiple targets
What does a MPLA probe consist of and what do they do?
Two oligonucleotides that recognise adjacent target sites on the DNA
What is MLPA used for?
To detect abnormal copy numbers at specific chromosomal locations
What do the two probes in MLPA detect?
One contains the sequence recognised by the forward primer
One contains the sequence recognised by the reverse primer
What has to happen before amplification can take place in MLPA?
Both probes hybridised and ligated into a complete probe
What happens after amplification in MLPA?
Fragment analysis of the product
What is next generation sequencing used for?
Enriching to sequence the only known disease genes relevant to the phenotype
What is the next generation sequencing method?
Target enrichment
Capture regions of interest with baits
Carry out hybridisation and column purification
What are the ethical considerations needed for exome and genome sequencing?
Inspect relevant genes first when analysing
Long patient consent process
Need some form of strategy for reporting incidental findings
What does the 100,000 genome project sequence?
Rare diseases- index cases and families
Cancer- germline and tumour samples
What does the 100,000 genome project do?
Brings direct benefit of whole genome sequencing and genetics to patients
What are tier 1 variants in the 100,000 genome project?
Known pathogenic,
protein truncating
What are tier 2 variants in the 100,000 genome project?
Protein altering (missense) Intronic (splice site)
What are tier 3 variants in the 100,000 genome project?
Loss- of-function variants in genes not on the disease gene panel
Where is most of the genetic testing in the UK done?
NHS diagnostic laboratory
What is the main role of the NHS diagnostic laboratory?
Help consultants reach a genetic diagnosis for patients to help guide their treatment and clinical management
What is clinical validity?
How well a test predicts the phenotype
What is clinical utility?
How well a test adds to the management of the patient
What are the options for the outcome of a diagnostic test?
Pathogenic mutation
Normal variation
Novel variant
How do you establish if a mutation is pathogenic?
Mode of inheritance
Genetic databases of published and unpublished data
Different types of mutagens
Missense/intronic mutation
