Clinical Genetics: Overview of genomic technologies in clinical diagnostics Flashcards
Give a brief overview of what PCR (polymerase chain reaction) is and how it works
- PCR is used to amplify a specific region of DNA, e.g. a gene or gene exon associated with a disease
- 3 stages: Denaturation, annealing and extension
- Each cycle doubles the amount of DNA copies of your target sequence
- Amplify enough DNA molecules so that we have sufficient material for downstream applications, e.g. sanger sequencing
What is fragment analysis?
- PCR followed by capillary electrophoresis which sizes the PCR product accurately
- Instead of being represented by bands as with gel electrophoresis the PCR products are represented by peaks
What can fragment analysis be used for?
- Can be used to detect repeat expansions or other small size changes in allele size (up to a few hundred bp)
- This is important because repeat expansions can cause diseases such as Huntingdon’s disease
What is Huntingdon’s disease?
- It is a severe neurodegenerative disorder caused by CAG repeat expansion in the Huntingtin (HTT) gene
How does the CAG expansion in the Huntingtin gene causes Huntingdon’s disease?
- Normal HTT gene has < 27 CAG copies; Intermediate 27-35 copies; Pathogenic > 35 copies
- Expanded protein produced from HTT gene with CAG repeat expansion is toxic and accumulates in neurons causing cell death
How is Huntingdon’s disease diagnosed?
- Diagnosed with fragment analysis
Give a brief overview of sanger sequencing
- Cycle Sequencing technique; based on the same principles as PCR
- Used to re-construct a nucleotide sequence
- Each of the 4 dideoxyribose nucleoside triphosphates (ddNTPs) in reaction mixture has a different dye so we can determine the nucleotide sequence.
- Can sequence up to 800bp of sequence per reaction so good for sequencing single exons of genes
What are some of the disadvantages of sanger sequencing?
- Slow
- Low-throughput
- Costly to perform for large numbers of samples
What can sanger sequencing be used for?
- Can be used to identify single nucleotide polymorphisms (SNPs), or mutations
Give a brief overview of Fluoresence in situ hybridisation (FISH)
- Used to microscopically detect large chromosomal abnormalities such as:
- Extra chromosomes
- Large deleted segments
- Translocations
How does fluoresence in situ hybridisation work?
- Design Fluorescent probe to a chromosomal region of interest
- Denature probe and target DNA
- Mix probe and target DNA together (hybridisation)
- Probe binds to the target DNA on the chromosome of interest
- Target fluoresces or lights up
Give a brief overview of Array CGH (comparative genomic hybridisation)
- Used for detection of sub-microscopic chromosomal abnormalities
- Patient DNA labelled Green
- Control DNA labelled Red
How does Array CGH work?
- Patient DNA and control DNA are extracted from samples
- Patient DNA labelled with Cy3, green and control DNA labelled with Cy5, red
- They are then mixed together and hybridised to the microarray
- Patient and control DNA compete to hybridise to the microarray
- Each spot on the array is then scanned to identify the colour of fluoresence it produces
What does each colour of fluoresence on the microarray for array CGH represent?
- No colour fluoresence = Equal hybridisation of patient and control DNA
- Red = More control DNA hybridised than patient DNA so there’s a loss of DNA in patient in that position in genome
- Green = More control DNA hybridised than control DNA so there’s a gain of DNA in patient in that position in genome
What is MLPA?
- Multiplex ligation-dependent probe amplification (MLPA)
- It’s a variation of PCR that permits amplification of multiple targets
- MLPA is used to detect abnormal copy numbers at specific chromosomal locations
- Can also detect sub-microscopic (small) gene deletions/partial gene deletions
- Usually used to see if there’s any abnormalities in a gene known to be involved in a specific condition