Overview Of Genomics Technologies In Clinical Diagnostics Flashcards
What is PCR and what is it used for?
Polymerase chain reaction is used to amplify a specific region of DNA
What are primers and what do they do?
Primers are complementary to regions we want to amplify in PCR. They flank the region.
What are + explain the 3 steps in PCR?
Denaturation - 90 degrees - high temp permits DNA strand separation
Annealing - 50 - 65 degrees - cooler temp allows primers to attach to ends of target sequence
Extension - 72 degrees - allows thermostable polymerase to add nucleotides at 3’ end of primer
What is fragment analysis?
PCR based assay
PCR followed by capillary electrophoresis - separates DNA in PCR product by size
What is fragment analysis used for?
detect repeat expansion or other small changes e.g. if more than one product present when only 1 in specific amount should be
diagnosing repeat expansion disease
Which disease is fragment analysis used for?
repeat expansion disease e.g. Huntington’s disease (neurodegenerative disorder)
What is Huntington’s cause by?
CAG repeat expansion on Huntington (HTT) gene
- above certain level pathogenic copies (protein produced is basic, too much protein produced - accumulates in neurons causing cell death)
What is sanger sequencing?
Cycle sequencing based off of PCR (sequences the PCR product)
What is the mechanism of sanger sequencing?
Each of 4 nucleotides has diff dye so can detect nucleotide sequence
What is sanger sequencing used for?
Sequencing single exons of genes (sequencing whole genome is costly)
- can identify SNPs or mutations (monogenic single gene mutations)
- detect mutations in family
What is the cause of cutaneous vasiculitis?
Mutation in gene C3
What is FISH?
Fluorescent in situ hybridisation
When can FISH be attached to cultured cells?
Metaphase spread so chromosomes are separated
What is the use of FISH and what does it detect? example
Detects large chromosomal abnormalities (can be seen under microscope)
- large deleted segments
- translocations
- extra chromosomes
e.g. trisomy 21 - down’s syndrome
What are the steps of FISH?
1) design fluorescent probe to chromosomal region of interest
2) denature probe and target DNA
3) Mix probe and target DNA (hybridisation)
4) Probe binds to target
5) Target flurosces/lights up