PCR and it's use in diagnostics Flashcards
What is PCR?
The polymerase chain reaction; a cyclical process to amplify specific segments of DNA using a thermostable DNA polymerase
What is a chain reaction?
A series of events
In which one event is dependent on it’s preceding event to sustain itself in order to occur
What typically happens in a chain reaction?
An exponential increase in the number of events that occur in the sequence
What allows for the specificity of amplifying segments of DNA?
The complementarity of the primers to the segment and the conditions (must be high stringency i.e. temperature at the Tm of the primer, so that only non-mismatched/ accurate copies form and not those from other parts of the genome)
What would happen if a PCR were performed at sub-optimal conditions?
Mismatches would be copied into the new duplex formed, so a non-specific product would be amplified
How many primers are required in PCR and where do they bind?
2, with each one binding to either side of the segment to be amplified (and so the primers must be complimentary to the ends of the segment)
How does DNA polymerase recognise where to bind? What forms as a result of binding? What happens as a result?
The binding of the primer with a single DNA strand forms a partially double stranded DNA. This is recognised by DNA polymerase where it binds and forms an initiation complex. The DNA polymerase proceeds to extend the double stranded molecule from the 3’ end of the non template strand/ 3’ end of primer
How does annealing occur during a PCR?
A DNA molecule is first denatured by heat to form 2 single strands. Then a primer (an oligonucleotide - short no. of nucleotides), complimentary to the start of the segment of DNA to be amplified, hybridizes with the DNA (forms H bonds - stabilizes it). The partially double stranded molecule is now a template for PCR and DNAP to add nucleotides to
Why do the single strands of DNA, following denaturation, bind with primers rather than renaturing?
A vast amount of primer is added to the mixture (with a small conc. of the template strand) which shifts the equilibrium to the side of hybridisation with the primer over renaturation to form the duplex. There is therefore competition present between the 2 states.
How would you go about amplifying RNA transcripts?
The DNA polymerase can only amplify DNA, so the RNA transcripts must convert to cDNA via reverse transcription before it can be amplified in PCR
What are the conditions required for PCR?
- A template strand
- A complimentary primer (20-30 BPs)
- Deoxy nucleotide triphosphates (dATP, dGTP, dCTP, dTTP)
- Mg2+ ions
- A roughly neutral pH
What are the 6 steps of a PCR reaction?
- Mix template, primer, enzymes and reactants together
- Heat mixture to 95 degrees; template duplex denatures and separates due to breakage of the H bonds
- Cool the mixture to the temperature of the primers Tm (for high stringency conditions in hybridisation) e.g. 55 degrees, allowing for the primers (2) to anneal to the template strand
- Increase the temperature to 72 degrees, optimal for the DNA polymerase to work
- DNA polymerase recognises the partial duplex, binds to form an initiation complex and adds nucleotides from the 3’ end of the primer
- Repeat the process again and again
What is a key property of DNA polymerase that allows it to survive the PCR reaction?
It is thermostable; it can retain activity upon repeated heating and cooling that would destroy most enzymes
Hence a polymerase from a thermophilic bacterium such as Thermus aquaticus is often used (Taq polymerase)
Why is there an exponential accumulation of product in PCR?
Every cycle results in a doubling of the product
What happens to the PCR reactions kinetics as the reaction goes on?
The reactants and primers deplete; acidification occurs since hydrogen ions are produced from dNTPs adding to the elongating strand; pyrophosphate is also produced; this results in a plateau of the graph since the env. in which the DNA polymerase works changes (and so no more product is formed)
