DNA PCR (Polymerase Chain Reaction) Flashcards
What does PCR Stand For?
Polymerase Chain reaction
What is a target region?
A short sequence of nucleotides called DNA primers.
These are synthesised DNA fragments that are identical to the DNA regions of interest.
What are the stages of a PCR Reaction?
Denaturation, Annealing, Extension.
What happens during the Denaturation stage of PCR?
Exposing DNA to higher temperatures, causes the hydrogen bonds between the bases which hold Double strands to break apart.
At the end of this process we are left with DNA in a single strand form ready for reforming.
What happens during the Annealing stage of PCR?
The temperature is dropped.
Primer from the primer mix recognises a site on the DNA, which flanks the target regions to be amplified, binding to the target regions.
What is a Primer?
A primer is a short synthetic piece of DNA that matches defined locations by complementary base pairing.
They are called primers as they mark the starting location of the synthesis of new DNA and prime the reaction.
What happens during the extension phase of PCR?
An enzyme called Taq Polymerase reads the template DNA adding complementary bases in the form of dNTP’s to the primer.
This results in a newly synthesised DNA strand.
What happens during a PCR cycle?
Step 1. The temperature is held between 90-95*C.
Step 2. The temperature is kept the same.
Step 3. The temperature is dropped to 55-65*C.
Step 4. The temperature is raised to 72*C.
Step 5. The temperature is kept the same.
Step 6. The temperature is dropped to 4*C.
Steps 2-4 are repeated for the number of cycles required.
What is Taq Polymerase?
A thermostable enzyme isolated from the heat-tolerant bacterium Thermus aquaticus.
What does Taq Polymerase do?
Allows for multiple cycles of amplification without needing any new enzyme being added.
Where does PCR occur?
A thermocycler
How does a thermocycler work for PCR?
Samples prepped for PCR with the correct dilution/ proportions of PCR reaction mix are placed in a thermocycler
This acts as a heating block which can precisely raise and lower the temperature as required.
Hydrolysis probes
Bind to the target DNA during annealing phase of PCR cycle.
Fluorophore quenching occurs via Forster/ fluorescence resonance energy transfer (FRET), from the fluorophore to a quencher molecule.
As the polymerase extends along the template strand of DNA exonuclease activity hydrolyses the hydrolysis probe.
Hydrolysis of the probe separates the fluorophore from the quencher preventing FRET.
The fluorophore then increases in fluorescence.
Hydrolysis probes used to detect each target are labelled with probes of different wavelengths.
This allows for independent measurements.
Dyes
FAM
- 84bp target
Cal fluor Gold 540 dye labelled probes
- 81bp and 163bp target amplicons on the Y chromosome
CXR
- Passive reference dye
TMR
- Internal Positive Control
- 435bp
- Indicates when inhibitors are present
Quasar 670
- Degradation index
- Provides a ratio of the DNA concentrations between the autosomal and the target.
Amplification plots
Exponential phase - All reaction components are in abundance, only RDS is the starting template DNA
Linear phase - One or more reaction components have been used up
Plateau phase - The reaction has stopped as all reaction components have been used
Y axis - Fluorescence unit
X axis - Number of PCR cycles
