15. Molecular Diagnostic Tools 2 Flashcards
What does FISH stand for?
Fluorescent in-situ hibridisation
What is FISH used for?
Detecting duplications or deletions of DNA smaller than detected by karyotyping
What are the steps in FISH?
Probes labelled with reporter molecules
Denature probe DNA
Probe is mixed with metaphase chromosomes on a slide
Screened for reporter molecules by fluorescence microscopy
What can FISH detect?
Translocations
Aneuploidy
Gain/loss of chromosomal region
What are the limitations of FISH?
Specific to what probes are designed for
Doesn’t detect small variations
Labour intensive
What does aCGH stand for?
Array-based Comparative Genomic Hybridisation
What is a-CGH used for?
Detection of aneuploidy and chromosomal copy number changes
What are the advantages of a-CGH?
Can detect aneuploidy and CNVs on a small scale
Whole genome scan
Less labour intensive
What are the steps in a-CGH?
Isolate DNA from patient and a control
DNA digestion
Fluorescently label patient and control different colours
Scanner picks up colour difference on microarray and gives ratio of how much DNA differs
What are the limitations of aCGH?
Does not detect balanced translocations
Difficult to distinguish SNPs with pathologically significant ones
What is array hibridisation used for?
Tells germline genotype of individual across many different SNPs
Identify carriers for single gene disorders
Identify risk variants for complex disease
What are the steps in array hibridisation?
- Genotyping array contains oligonucleotides designed to target SNPs of interest
- Bound to silica beads spread across microarray
- DNA fragmented and added to array, bases are labelled with fluorescent tags
- Fragments hibridise to oligonucleotides based on Watson-Crick model
- Single base-pair extension is conducted
- Genotype is read based on colour
What are the benefits of next generation DNA sequencing?
Detects germline and somatic variation
Detects all variation: both large and small scale
