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
use of mRNA in qPCR
- mRNA is an indicator for gene expression.
- can only use DNA (not RNA) as a template in a PCR.
- mRNA is turned into so-called cDNA using an enzyme called Reverse Transcriptase.
Synthesis of cDNA
1.Poly-dT primers are used, because they anneal to the 3’ end of nearly all eukaryotic mRNAs (due to the presence of polyA-tail)
2.Reverse Transcriptase extends that primer and reverse transcribes RNA into DNA. This gives you an RNA/DNA hybrid helix.
3. RNA strand is degraded with RNAse H and a complementary DNA strand is synthesised with DNA polymerase producing a ds DNA copy of the original mRNA.
How does the qPCR cycler measures the amount of DNA that is present after each PCR cycle in real time
by using an intercalating DNA dye or a fluorescent probe
Ct value =
- the value where the PCR curve crosses the threshold.
- this is the value that is used for the analysis.
- The higher the Ct (30-35), the less the mRNA detected is present.
How do you measure the relative expression
∆Ct value (CT housekeeping – CT GOI).
How do you express the change in expression level of the GOI?
∆∆Ct (∆Ct stimulated - ∆Ct unstimulated).
RT-PCR for SARS-CoV-2 diagnosis
- extract RNA from nasopharyngeal swabs (SARS-COV-2 has an RNA genome)
- This is then converted into cDNA for use in RT-PCR
- Specific primers for SARS-COV-2 genes are used in theRT-PCR (usually ‘’multiplexed‘’, so 2-3 genes are detected at the same time)
What are the advantages of using Northern Blotting to quantify mRNA transcript levels
- Can determine size of the RNA transcript and could pick up alternative transcripts
- Control house keeping gene B-actin mRNA doesn’t change during stimulation, therefore this normalises for total RNA level in sample.
What are the disadvantages of using Northern Blotting to quantify mRNA transcript levels
-Technicallymore difficult
-Not as quantitative
Microarrays allow more…
global gene expression analysis
microarray process
- DNA oligonucleotides corresponding to specific genes, are immobilised on a small glass, silicon or nylon slide. Each field contains many copies of a single stranded DNA probe.
- mRNA is extracted from cells, converted to cDNA and labelled with a fluorescent dye.
- The chip is exposed to the labelled cDNA
- The array is scanned to measure fluorescence intensity. The location of the bound sample on the array is detected and gene expression is determined.
what is an example of microarray applications
Cancer Biopsy to normal tissue sample (Stimulated samples compared to non- stimulated control.)
benefits of RNAseq
- uses Next Generation Sequencing technology on cDNA libraries generated from RNA isolated from cells.
- Generates unbiased whole transcriptome data
- Can identify novel splice variants and non-coding RNAs
