Overview of genomic technologies in clinical diagnostics Flashcards
What is PCR?
Three step process
- Denature template DNA
- Anneal the primers
- Extension step, creating new DNA molecules and we;ve doubled the amount of DNA in a cycle
Used to
- Amplify a specific region of DNA - primers flank the region you want to amplify
Repeated 30-40 times.
How do you analyse the PCR product by fragment analysis?
- PCR based assay
- PCR followed by capillary electrophoresis, separate out DNA molecules in our PCR product by size.
- Use this method to detect changes in size in PCR fragments and to detect repeat expansions
- Can be used to detect repeat expansions or other small size changes (up to a few hundred bp)
Huntington’s disease is diagnosed with fragment analysis, what is Huntington’s disease?
A repeat expansion disease, severe neurodegenerative disorder.
Caused by CAG repeat expansion in Huntingtin (HTT) gene.
Expanded protein is toxic and accumulates in neurons causing cell death.
What does sanger sequencing of PCR products identify?
- Each of the 4 DNA nucleotides has a different dye so we can determine the nucleotide sequence
- Generate up to 800bp of sequence per sanger sequencing reaction, good for sequencing single exons of genes
- Slow, low throughput and costly to perform large numbers of samples
Sanger sequencing is used to identify SNPs or mutations by determining the nucleotide sequence by using the dyes.
How is fluorescent in situ hybridisation 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 on DNA sample
- Target fluoresces or lights up so we can see the target
cultured cells from patients are spread during metaphase to tease out chromosomes + probe them with FISH
What is FISH used to detect?
- To detect large chromosomal abnormalities
- Detect microscropic (5-10Mb) chromosomal abnormalities, 5-10mil bp
- Can detect extra chromosomes
- Can look for large deleted segments
- Can look for translocations where we have pieces of chromosomes moving from one place to another
What is Array comparative genomic hybridisation (CGH) used for?
- Detection of sub-microscopic chromosomal abnormalities
- Patient DNA labelled green
- Control DNA labelled red
How is Array-CGH carried out?
- Patient and Control DNA labelled green and red
- Mixed together and hybridised to a microarray
- signal, green or red detected
- equal hybridisation = no net red or green dye = no net loss or gain
- Increased green signalling = gain
- Increased red = loss
What is multiplex ligation-dependent probe amplification?
- MLPA is a variation of PCR that permits amplification of multiple targets
- Uses probes, each probe consists of two oligonucleotides which recognise adjacent target sites on DNA
- One probe oligonucleotide has sequence recognised by forward primer, other sequence recognised by reverse
- When both are hybridised to their targets they can be ligated into a complete probe
What is the three steps in MLPA?
- Hybridising the two probes to the denatured template DNA, which is more likely a patient DNA sample
- Ligation, ligate and join together forward and reverse probes
- PCR amplification of our probe to generate an amplified library or product of our target.
What is performed after on the MLPA product to determine dosage of MLPA product and its size?
Perform fragment analysis (Capillary electrophoresis) of MLPA product to determine dosage and size.
What is an important use of MLPA?
- To determine relative ploidy (how many chromosome copies) at specific locations
- For example probes may be designed to target various regions of chromosomes of a human cell
- 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 are the benefits of using exome sequencing?
- 21,000 genes genome, often only interested in gene protein coding exons/exome, which is 1-2% of the genome
- 80% of pathogens are protein coding
- more efficient to seqeunce only exome and not entire genome
- Cheaper, £200-300 compared to £1000
What challenges are there with using exome and genome sequencing?
Interpreting results.
- 20,000 genetic variants identified per coding genes exome
- 3 million variants in whole huamn genome, challenging to find pathogenic mutation
Challenging to implement sequencing
Infrastructure and training (IT and clinical scientists) needed to perform sequencing and interpret data.
What are some ethical considerations to consider with sequencing?
- Modified patient consent process. Patients need to consent to have whole genome sequenced
- Data analysis pathways – inspect relevant genes first, don’t want to look at places we don’t need to
- Consider strategy for reporting incidental findings e.g a mutation which predisposes to another disease that hasn’t been consented for
