Overview of Genomic technologies in Clinical diagnosis Flashcards
What are some different genomic 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 PCR
- PCR is used to amplify a specific region of DNA
- Design specific primers which are oligonucleotide sequences =complimentary to amplicon
- Primers flank the region you want to amplify.
- Each cycle doubles the amount of DNA copies of your target sequence
- Amplify enough DNA molecules = have sufficient material for downstream applications
What are the three steps of PCR -
Denaturation -High temperature permits separation of DNA strands
Annealing- Cooler temperature allows primer to attach to ends of target sequence
Extension-Temperature allowing thermostable polymerase to add nucleotides at the 3’ end of primers
What is Capillary electrophoresis
PCR based assay
Following PCR we carry out capillary electrophoresis
(This is a technique which separates ions based on electrophoretic mobility )
-We size the PCR product
-This can be used to detect repeat expansions or small size changes (up to a few hundred bp)
What is a repeat expansion disorder ?
Genetic diseases that are caused by expansions in DNA repeats.
Example- Huntington’s disease (severe neurodegenerative disorder )
Cause -CAG repeat expansion in the HTT gene
Normal - less than 27 copies
Intermediate-27-35
Pathogenic-35 copies
Expanded protein is toxic and accumulates in neurons causing cell death
Can be diagnosed using capillary electrophoresis
Describe Sanger sequencing
- Each of the 4 DNA nucleotides has a different dye so we can determine the nucleotide sequence.
- Up to 800bp of sequence per reaction
- Good for sequencing single exons of genes
- Slow, low-throughput and costly to perform for large numbers of samples
What can sanger sequencing be used for?
We can identify single nucleotide polymorphisms (SNPs), or mutations
Describe FISH (Fluorescent in situ hybridisation)
It is used to detect (Microscopic) large chromosomal abnormalities
Cells from patients spread during metaphase
Can look for:
•Extra chromosomes
•Large deleted segments
•Translocations(chunks of chromosomes moving from one place to another)
How does FISH work?
- Design Fluorescent probe to chromosomal region of interest
- Denature probe and target DNA
- Mix probe and target DNA (hybridisation)
- Probe binds to target
- Target fluoresces or lights up so we can see our target
What type of observations can we make using FISH
Outline two ways FISH can be used
Special Karyotyping
-pairs of chromosomes are manipulated to have distinctive colours.
makes it easier to detect chromosomal abnormalities
(Trisomy 21)-Three copies of 21 -Down syndrome
Target specific
-Probe for specific chromosomes
What is Array CGH (comparative genomic hybridisation )
Used to detect sub-microscopic chromosomal abnormalities .
Patients DNA labelled Green
Control DNA labelled Red
Can see patient sample whether it has net loss/gain
Increased green signal will show a gain
Describe MLPA
Multiplex ligation-dependent probe amplification
- Variation of PCR that permits amplification of multiple targets
- Probe= two oligonucleotides -recognise adjacent target sits on DNA
What can MLPA be used to detect?
Use:
-To detect abnormal copy numbers at specific chromosomal locations
-Can detect sub-microscopic gene deletions/partial gene deletions
How does MLPA work?
Three step process
- Hybridisation
- Two probes are hybridised (forward/reverse primer) - Ligation
- Join together the two probes
3.Amplification
PCR amplification of the two probes to generate an amplified library
How can we analyse following MLPA?
Using capillary electrophoresis
(fragment analysis)
-Used to determine ploidy(how many chromosome copies)-specific locations
-Signal strength of the probes is compared to those obtained from reference DNA sample
(contains two copies of the chromosome )