Overview of Genomic Technologies in Clinical Diagnostics Flashcards
What are the genomic technologies currently available?
- PCR
- Fragment analysis
- Sanger Sequencing
- Fluorescence in situ hybridisation (FISH)
- Array comparative genomic hybridization (Array CGH)
- Multiplex ligation-dependent probe amplification (MLPA)
- Next Generation sequencing
What is PCR?
Enzyme based method used to amplify segments of DNA using a thermal polymerase in a cyclical process
When is PCR used?
Fundamental for many DNA applications
PCR used to amplify specific regions of DNA
Primers flank the region you want to amplify
What is fragment analysis?
PCR based assay
PCR followed by capillary electrophoresis (bands appear as peaks)
What is fragment analysis used for?
To detect repeat expansions or other small size changes (up to a few hundred bp)
Give an example of a repeat expansion disease identified using fragment analysis
Huntington’s disease – severe neurodegenerative disorder
What causes Huntingtons disease?
Caused by CAG repeat expansion in the Huntingtin (HTT) gene
Expanded protein is toxic and accumulates in neurons causing cell death
Describe the normal and pathogenic range of CAG repeats
Normal < 27 copies; Intermediate 27-35 copies; Pathogenic > 35 copies
What is sanger sequencing?
Cycle Sequencing; based on the same principles as PCR
How does sanger sequencing identify base sequences?
Each of the 4 DNA nucleotides has a different dye so we can determine the nucleotide sequence
What is sanger sequencing used for?
Good for sequencing single exons of genes
Identify SNPs
Up to 800bp of sequence per reaction
What are the downfalls of Sanger sequencing?
Slow, low-throughput and costly to perform for large numbers of samples
Outline how sanger sequencing is carried out
- Sample placed on a plate
- Sample taken up into capillaries
- Passed through a laser
- 4 bases show different colours
What is FISH?
Fluorescent in situ hybridisation
- Cultured cells, metaphase spread
- Microscopic (5-10Mb)
What can we detect using FISH?
Large chromosomal abnormalities
- Extra chromosomes
- Large deleted segments
- Translocations
Outline how FISH is carried out
- Design Fluorescent probe to chromosomal region of
interest - Denature probe and target DNA
- Mix probe and target DNA (hybridisation)
- Probe binds to complementary target sequence
- Target fluoresces or lights up
How is FISH used for special karyotyping?
Designed lots of probes to mark all chromosomes in a different way to colour coordinate them (barcode)
What is array cgh?
Array comparative genomic hybridisation is for detection of submicroscopic chromosomal abnormalities as well as large
What is the process of array CGH
- Patient DNA labelled Green
- Control DNA labelled Red
- Mix DNA samples together
- Hybridise sample to array microchip w/ spots covering
whole genome on - Patient DNA and control DNA bind to spots
Describe array cgh results
Increased green signal over a chromosomal segment in patient DNA indicates a gain in the patient sample not present in parents
Red indicates a loss in DNA
What is array CGH used for?
Can identify someone’s copy no. status
- Good for analysing pathogenic chromosomal mutations
- Shows structural variety
What is MLPA?
Multiplex ligation-dependent probe amplification (MLPA) is a variation of PCR that permits amplification of multiple targets
What does each MLPA consist of?
Each probe consists of two oligonucleotides which recognize adjacent target sites on the DNA
What can MPLA identify?
MLPA used to detect abnormal copy numbers at specific chromosomal locations
and sub-microscopic (small) gene deletions/partial gene deletions
What does each MLPA probe contain?
One probe oligonucleotide contains the sequence recognized by forward primer
The other contains the sequence recognized by reverse primer
In MLPA when do the 2 separate probes become one probe?
Only when both probe oligonucleotides are hybridized to their respective targets, can they be ligated into a complete probe
How do we amplify and visualise the complete probe in MLPA?
Perform fragment analysis (capillary electrophoresis) of MLPA product
Describe a use of MLPA
An important use of MLPA is to determine relative ploidy (how many chromosome copies?) as specific locations
How can we determine the strength of the probe signals in MLPA?
The signal strengths of the probes are compared with those obtained from a reference DNA sample known to have two copies of the chromosome
What is a commonly used NGS?
Solexa sequencing-by-synthesis (SBS) developed end of 2005
Sequencing market is now dominated by Illumina SBS sequencing
What are the benefits of NGS?
NGS is an end to sequential testing - Wider range of tests in a shorter time for less money
What are the current strategies and aims of NGS?
Disease panels
- Enriching to sequence only known disease genes
relevant to phenotype - Panels expandable to include new genes as they are
published - Potentially pathogenic variants confirmed by Sanger
sequencing
How many genes are present in the human genome?
There are ~21,000 genes in the human genome
How significant is the exome?
Gene protein coding exons or ‘exome’ represents 1-2% of genome
Some ~80% pathogenic mutations are protein coding
What is the advantages of exome sequencing?
More efficient to only sequence bits we are interested in, rather than entire genome
Costs £1,000 for a genome, but only £200-£300 for an exome
What are the uses of exome sequencing?
- Target enrichment
- Capture target regions of interest with baits
- Potential to capture several Mb genomic regions
(typically 30-60 Mb)
What is the future of whole genome sequencing WGS?
Universally accepted that genome sequencing will become commonplace in diagnostic genetics
What other tests are available other than WGS?
Panels/single gene tests
- May be more suitable for diseases, e.g. cystic fibrosis
Capillary-based methods
- Repeat expansions, MLPA, family mutation confirmation
Sanger sequencing
Array-CGH
- large sized chromosomal aberrations
What is the significance of WGS?
Whole genome sequencing is NOT trivial
Interpretation of clinical genomes is currently has a substantial manual component
What is the biggest problem with WGS and WES?
Result interpretation is the greatest challenge
- 20,000 variants per coding genes ‘exome’
- 3 million variants in a whole human genome
What are the ethical considerations of WES and WGS?
- Modified patient consent process
- Data analysis pathways – inspect relevant genes first
- Strategy for reporting ‘incidental’ findings
What is the NHS Diagnostic Library?
Accredited laboratory: ISO standard 15189 for Medical Laboratories
Has scientific, technical and administrative staff
What are the roles of the NHS diagnostic library?
Provide clinical and laboratory diagnosis for genetic disorders
Liaise with clinicians, nurses and other health professionals
Provide genetic advice for sample referrals and results
What is the main role of the NHS Diagnostic library?
To help Consultants reach a genetic diagnosis for individuals and families to help guide treatment and clinical management
How do consultants reach a genetic diagnosis?
Perform specific tests with proven:
Clinical Validity
- How well the test predicts the phenotype
Clinical Utility
- How the test adds to patient management
Outline the diagnostic test outcomes
- Pathogenic mutation
- Normal variation; Polymorphism
- Novel variant ; Investigations to establish significance
How do we interpret diagnostic test results?
To establish if a mutation is pathogenic figure out the following:
- Mode of inheritance
- Locus-specific databases of (un)/published data
- Nonsense, frameshift, splice site (exon +/-2 bp)
mutations
- Missense/intronic mutation
- In-silico tools for missense + splicing mutations
