PCR Flashcards
What does PCR stand for?
Polymerase Chain Reaction
What does PCR do?
It allows the amplification of a DNA fragment using a DNA template
Why is PCR useful?
- Required for analysing sequences (such as detecting species-specific genes, species IDing using molecular markers, and identifying age and molecular phyologeny)
- Required for the identification of pathogens in medicine
- Used for recombinant systems (combining different pieces)
- Forensics, paternity test…
How does PCR work (simple description)?
It is the process of synthesising a new complementary DNA strand (to a template) using DNA polymerase to add nucleotides.
Deoxyribonucleotides are sequentially added to the 3’-OH ends of a polynucleotide chain.
Describe the stages and process of PCR (in full)
- Denaturation (this is the separation of the DNA strands): carried out by heating the DNA to 94-96 degrees Celsius to break apart the hydrogen bonds between the bases of complementary DNA strands.
- Cycling: this involves: denaturation (strand separation), annealing (primer binding) and elongation
Denaturation is carried out as above. Primers are added which bind to the complementary part of the template strand. Primers are composed of forward and reverse strands which break apart once the melting temperature of the primer is reached (this is known as Tm and it generally between 50-55 degrees Celsius). The primer is a short strand to which the DNA polymerase binds to extend the sequence. - Extension
DNA polymerase extends the primers, producing two double strands (from a single double strand). This is normally carried out at 68-72 degrees Celsius.
This can be repeated many times, producing target double strands with an exponential increase - normally this is carried out at least 30 times. Eventually there is a loss of components and the increase in strands produced plateaus.
Note that the first denaturation occurs for several minutes, before cycling for around 30-40 cycles, and a final longer elongation period.
What dictates the temperature at which annealing is carried out in PCR?
This is the melting temperature of the DNA primers.
This can be predicted based on the number of hydrogen bonds present (more bonds = more energy required so higher temperatures) and generally sits between 60-65 degrees Celsius.
This temperature is low enough that the primers will anneal to the exact sequences. Too high and the primers won’t anneal, and too low they begin to anneal to sequences which are similar but are not exact.
This temperature is also normally just below the melting temperature of the primer (this is lower than the melting temperature of the DNA strand as there are fewer hydrogen bonds) to reduce the likelihood of the reverse and forward primer strands binding back together.
Describe the DNA primers used in PCR.
- The DNA primers are added as primer DNA pairs. They are composed of forward and reverse primers, which bind to complementary DNA sequences (allowing one DNA double strand to form two new double strands at the end of PCR).
- These primers are selected as they match the 5’ and 3’ end of the target sequence.
- In general they are around 18-30 nucleotides long
What is the DNA polymerase used?
A specific thermostable DNA polymerase is used which can still operate at temperatures of up to 68-72 degrees Celsius and survive the heating in the earlier stages.
The temperature of elongation is dependent on the DNA polymerase used and its optimum activity temperature
What equipment is required for PCR analysis?
- Gloves and pipettes/tips
- Ice (keeps components inactive before PCR begins)
- Microtubes/eppendorf reaction tubes (have a little lid for reactions to occur in)
- PCR tubes
- PCR machine (the machine controls the temperature throughout the process)
- Gel electrophoresis
- Risk assessment (COSHH forms for ethidium bromide use - this is a carcinogen)
What is carried out to test the success of PCR?
Gel electrophoresis (ensures the correct DNA has been amplified)
Which reaction components are required for PCR?
- DNA polymerase (this needs to be thermostable)
- Specific primer DNA pairs including forward and reverse (specific to the 3’ and 5’ ends of the target sequence)
- Nucleotides (dATP, dGTP, dCTP, dTTP)
- The target DNA template
- DNA polymerase reaction buffer (mimics cellular environments for optimal function)
- H2O (all living systems work in a liquid environment)
- Mg2+ (this is a cofactor for the function of DNA polymerase)
What can the target DNA sequence for PCR be?
- Genomic DNA
- cDNA (copy DNA)
- Plasmid DNA
- A library (a collection of DNA molecules)
Which constituents are added to specifically support the functioning of DNA polymerase?
- Mg2+ (a cofactor for DNA polymerase)
- DNA polymerase reaction buffer
- H2O (all living systems work in a living environment)
What are two examples of DNA polymerase used in PCR and where were they found? Compare the two.
- taq polymerase (found in thermophilic bacteria thermus aquaticus, isolated from hot springs in Yellowstone National Park)
- Pfu polymerase (from the bacteria Pyrococcus furiosus, found in marine hot vents)
Pfu polymerase is slower at elongation but more accurate as it carries out proof reading (this is achieved via 3’ - 5’ exonuclease, which removes misincorporated nucloetides).
Therefore:
- Use Taq polymerase for processivity
- Use pfu polymerase for fidelity
How do you calculate the melting temperature of primers?
Tm = 2(A+T) + 4(C+G)
