Genetic Technologies Flashcards
What is PCR?
Amplifying a specific known reigon of DNA
primers are used to flank the region you want to amplify
Each cycle doubles the amount of DNA copies of your target sequence
Amplify enough DNA molecules so that we have sufficient sample material
What is fragment analysis?
A PCR based assay
PCR followed by capillary electrophoresis
Here we are sizing the PCR product to detect repeat expansions or other small size changes
What is Huntington’s disease?
- Server neurodegenerative disorder
- caused by CAG repeat expansion in the Huntington Gene
- pathogenic when there are MORE THEN 35 copies
- expanded protein is toxic (accumulates in neurones causing cell death)
- Diagnosed with FRAGMENT ANALYSIS
What is Sanger sequencing?
- cycle sequencing
- different dye for each four nucleotides
- reading dyes to obtain DNA sequence
- single nucleotide polymorphisms (SNPs) or mutations are identified
What is FISH (fluorescent in situ hybridisation)?
- using cells from patients and using a metaphase spread
- to detect large microscopic abnormalities
- detect large deleted segments
- detect translocations
How is FISH carried out?
- Design fluorescent probe to chromosomal region of interest
- Denature probe and target DNA
- Mix probe and target DNA (hybridisation)
- Probe binds to target
- Target fluoresces
What is Array CGH (comparative genomic hybridisation) used for?
- for detection of sub-microscopic chromosomal abnormalities
Patient DNA - green
Control DNA - red
What occurs during Array CGH?
2 florescently labelled samples are mixed together
They hybridise to the microarray
Microarray scanner measures fluorescent signals
Computer software analyses the data and generates a plot
Increased green signal over a chromosomal segment in the patient DNA indicates a gain in the patient sample not present in the parents.
What is MLPA.
Multiplex ligand dependant amplification
Variation of PCR that permits amplification of multiple targets
Used to detect abnormal copy numbers at specific chromosomal locations
Can detect sub microscopic gene duplications/ partial gene deletions
How does MLPA function?
Two primers are used and detect different oligonucleotide sequences (forward primer and reverse primer)
Only when BOTH OLIGONUCLEOTIDES are hybridised to their respective targets can they be ligated into a complete probe
Amplified target
Then fragment analysis of MLPA to determine relative ploidy (how many chromosome copies?) at specific location
The signal strength of the probes are compared with those obtained form a reference DNA sample known to have two copies of the chromosomes
What is whole exome sequencing?
- used for identifying disease causing genes
- only interested in the protein coding exons (more efficient)
- cheaper
How is whole exome sequencing carried out?
By target enrichment
Capture target regions of interest with baits (biotinylated RNA library)
Potential to capture several Mb genomic regions
Why wouldn’t all tests automatically move to whole genome sequencing?
- panels/single gene tests may still be more suitable for some diseases
- capillary based methods: repeat expansions, MLPA, family mutation confirmation Sanger sequencing
- Array-CGH: large sized chromosomal aberrations
What are the limitations of Exome and Genome sequencing?
Result interpretation is the greatest challange
- large numbers of genetic variants are detected at once
Ethical considerations
- modified patient consent process
- data analysis pathways (analyse relevant genes first)
- strategy for reporting “incidental” findings
Infrastructure and training (time consuming to manually do)
What is the 100,000 genome Prodject?
Being direct benefit of WGS and genetics to patient
Enable new scientific discovery and medical insights in disease - eg. Personalised medicine
Genomes are collected from genomic medicine genders
In rare diseases - index cases + families
Cancer - germaline (blood) and tumour samples
What are the three tiers the panel is divided into?
Tier 1:
- known pathogenic
- protein truncating (mechanism known)
Tier 2:
- protein altering
- intronic
Teir 3:
- Loss of function in genes not on the disease gene panel
What role does the NHS diagnostic laboratory have?
Help consultants reach a genetic diagnosis for individuals and families to help guide treatment and clinical management.
Perform specific tests with:
- clinical validity: how well the test predicts the phenotype
- clinical utility: how the test adds to the management of the patient
UKGTN approved tests
What kind of testing does the NHS laboratory do?
Diagnostic
Predictive
Carrier(recessive)
Informed consent - genetic counselling (implications for other family members)
Diagnostic testing is available for all consultant referrals
What are the the 3 diagnostic test outcomes?
Pathogenic mutation
Normal variation - polymorphism
Novel variant - investigate clinical significance
How do we establish if a mutation is pathogenic?
- mode of inheritance
- genetic databases of published and unpublished data
- nonsense, frameshift, splice site
- missense/intronic mutation
What are the advantages and disadvantages of Sanger sequencing?
Advantages:
Up to 800bp per sequence reaction (good for single exons of genes)
Disadvantages:
Slow
Low-throughput
Costly to perform on large numbers of samples
