Overview of Genomic Technologies in Clinical Diagnostics Flashcards
List some genetic technologies.
- 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
Describe Polymerase Chain Reaction (PCR).
It is fundamental for any DNA application.
PCR is used to amplify a specific region of DNA; primers flank the region you want to amplify.
Each cycle doubles the amount of DNA copies of your target sequence
We amplify enough DNA molecules so that we have sufficient material to sequence or for other applications.
How would you perform fragment analysis after PCR?
PCR is followed by capillary electrophoresis.
Here, we are sizing the PCR product.
This can be used to detect repeat expansions or other small size changes (up to a few hundred basepairs).
Give an example of a repeat expansion disease that was diagnosed using fragment analysis.
Huntington’s disease is a severe neurodegenerative disorder.
It’s caused by CAG repeat expansion in the Huntingtin (HTT) gene.
- Normal < 27 copies;
- Intermediate 27-35;
- Pathogenic > 35
The expanded protein is toxic and accumulates in neurons, causing cell death.
This was diagnosed with fragment analysis.
Briefly, describe Sanger sequencing.
It goes through cycle sequencing; it’s based on the same principles as PCR.
Each of the 4 DNA nucleotides has a different dye so we can determine the nucleotide sequence.
Here, we are reading the dyes to obtain the DNA sequence. We can identify single nucleotide polymorphisms (SNPs),or mutations in this way.
We can sequence up to 800 basepairs of sequence per reaction.
It is accurate (99.99% of the time).
In one reaction, you sequence one sequence.
However, it is slow and has a low-throughput.
It is also costly to perform.
What does FISH stand for, and what would you use it for?
It stands for Fluorescence In Situ Hybridization.
We can use it to detect large chromosomal abnormalities.
Examples of such abnormalities include:
- Extra chromosomes
- Large deleted segments
- Translocations
How would you perform FISH?
- Design a fluorescent probe to the chromosomal region of interest.
- Denature the probe and target DNA.
- Mix the probe and target DNA together (hybridisation).
- The probe binds to the target.
- The target now fluoresces or lights up.
What is Array CGH, and what is it used for?
It is an array of comparative genomic hybridisation.
It is used for the detection of sub-microscopic chromosomal abnormalities.
The patient DNA is labelled green. while the control DNA is labelled red.
Describe how to perform an array CGH.
STEPS 1-3: The patient and control DNA are labelleed with fluorescent dyes and applied to the microarray.
STEP 4: The patient and control DNA compete to attach, or hybridize, to the microarray.
STEP 5: The microarray scanner measures the fluorescent signals.
STEP 6: The computer software analyses the data and generates a plot.
The plot shows a patient array comparative hybridisation profile.
An increased green signal over a chormosomal segemnt in the patient DNA indicates a gain in the patient sample that is not present in the parents.
What is MLPA?
Multiplex ligation-dependent probe amplification (MLPA) is a variation of PCR that permits amplification of multiple targets with only a single primer pair.
Each probe consists of two oligonucleotides which recognize adjacent target sites on the DNA.
One probe oligonucleotide contains the sequence recognized by the forward primer, the other contains the sequence recognized by the reverse primer.
Only when both probe oligonucleotides are hybridized to their respective targets can they be ligated into a complete probe.
What do we use MLPA for?
We use MLPA to detect abnormal copy numbers at specific chromosomal locations.
MLPA can detect sub-microscopic (small) gene deletions/partial gene deletions.
We perform fragment analysis of MLPA products.
An important use of MLPA is to determine relative ploidy (how many chromosome copies?) at specific locations.
For example, probes may be designed to target various regions of chromosome of a human cell.
The signal strengths of the probes are then compared with those obtained from a reference DNA sample known to have two copies of the chromosome.
Describe the advancements in the technology of Next Generation Sequencing.
Technological advances since the end of the human genome project have brought about a decrease in the cost of DNA sequencing.
Development of new NGS methods began 13 years ago with 454 pyrosequencing.
DNA sequencing throughout has jumped 10 orders of magnitude.
Solexa sequencing-by-synthesis (SBS) was developed at the end of 2005.
The sequencing market is now dominated by Illumina SBS sequencing.
Describe exome sequencing.
There are ~21,000 genes in the human genome.
Often we are only interested in the gene protein coding exons or ‘exome’, which represents 1-2% of the genome.
Some ~80% of pathogenic mutations are protein coding.
hence, it is more efficient to only sequence the bits we are interested in, rather than the entire genome.
It costs £1,000 for a genome, but only £200-£300 for an exome.
You would use target enrichment. You capture target regions of interest with baits.
With exome sequencing, there is the potential to capture several Mb genomic regions (typically 30-60 Mb).
What are some drawbacks of exome and genome sequencing?
Result interpretation is the greatest challenge:
- 20,000 variants per coding genes ‘exome’
- need for good variant databases of well phenotyped cases/control sequences
Ethical considerations
- modified patient consent process
- data analysis pathways – inspect relevant genes first
- strategy for reporting ‘incidental’ findings
Infrastructure and training (particularly IT)
What does the NHS Diagnostic Laboratory do?
The main role of the lab is to help consultants reach a genetic diagnosis for individuals and families to help guide treatment and management.
Perform specific tests with proven:
- Clinical Validity: how well the test predicts the phenotype
- Clinical Utility: how the test adds to the management of the patient
They perform UKGTN (UK genetic testing network)-approved tests.
They have in-depth and up-to-date knowledge of the genetic diseases covered.
They perform translational research.
