PCR and it's Application Lecture 5 and 6 Flashcards
Polymerase chain reaction (PCR) overview
- invented by Kary Mullis in 1985-87
-working at Cetus Corporation, synthesising oligonucleotides for study of mutations in sickle cell anemia - Problem: limited sample (human genomic DNA)
- Nobel prize in Chemistry, 1993
-Hoffmann-La Roche bought licence off Cetus for $300 million.
The polymerase chain reaction (PCR) allows us to…
amplify and isolate a DNA sequence that we are interested in.
What principle is PCR based on?
It is based on the principle of natural DNA replication carried out by DNA polymerase in cells.
Using purified/recombinant DNA polymerase, where is semi-conservative amplification of DNA carried out?
in a test tube.
How are only selected parts of the DNA is amplified
By using primers
Natural DNA replication starts from a short RNA primer that the DNA polymerase extends at the
3’ end
Initial (historic) experimental set-up:
- E. Coli DNA polymerase was not heat-stable, had to be added in fresh after each denaturation step.
Now we have heat-stable polymerases - ‘Cycling’, i.e. change in temperature had to be done manually by moving the tubes into water baths of different temperatures
Taq DNA polymerase properties
*isolated from the bacterium Thermus aquaticus, which lives in hot springs,
* heat-stable DNA polymerase, which survives the denaturation step and elongates DNA at 72oC.
* first published in 1976
* Science ‘Molecule of the Year’ 1989
What is other heat-stable polymerase are there
Pfu and Vent, isolated from other thermophiles.
What is in a Typical PCR reaction:
H2O
Buffer
MgCl
dNTPs
Template to be amplified
2 Primers (forward and reverse)
Taq polymerase
Denaturation
- 95°C
- separation of double strands
primer annealing
- 50-68°C
- depends on melting temperature of the primers
elongation.
72°C
Primer design considerations
- need to frame (flank) the sequence that you want to amplify.
- should have at least 15bp long complementary sequence to template
- Forward and reverse primer should have a similar annealing temperature.
- Primers should not form strong primer dimers or have extensive secondary structures.
What does annealing temperature depend on
annealing temperature depends on length and GC content. Usually, annealing temperatures should be between 50 and 70 ̊C.
what is PCR used for in Medicine
diagnostics for viral infections (HIV), for genetic disorders, HLA typing before transplantation
what is PCR used for in Forensics
amplify DNA from tiny amounts of blood, tissue, semen, DNA fingerprinting, paternity tests
what is PCR used for in Evolutionary studies
molecular phylogenetics, amplify ancient DNA, e.g. from Woolly Mammoth
How is DNA fingerprinting by PCR based on length polymorphisms carried out
Analyses Microsatellite regions. With PCR primers flanking the microsatellite region, the microsatellites can be amplified and their size compared on a gel.
what are Microsatellite regions?
variable number of tandem repeats, (VNTR): short sequences, e.g. CA, repeated 4-30 times
actual DNA fingerprint analysis
An actual DNA fingerprint analysis would analyse several different VNTR loci to reliably differentiate individuals
Real-time PCR
In a Real-time PCR, we measure the DNA amplification (the amount of DNA in the tube) in ‘real- time’ after each cycle.
what is the aim in Quantitative Real-time PCR
quantify the amount of template DNA (starting material).
what is qPCR used for
gene expression studies
