PCR and its role in diagnostics Flashcards
What type of method is PCR?
An enzyme based method to specifically amplify segments of DNA using a thermal DNA polymerase in a cyclical process
What is the definition of a chain reaction?
Series of events in which each one of which is dependent upon the preceding event to sustain itself
Where does specificity stem from in PCR?
Specificity stems from complementarity of primers
When is PCR method only specific and what does this prevent?
Specific only if annealing is undertaken at melting temperature Tm of the primers
Prevents mismatched base pairing
What polymerase is used in PCR?
DNA dependent DNA polymerase is used
What does DNA polymerase recognise?
Recognises a specific structure consisting of a partially double stranded DNA forming an initiation complex with it
What does annealing result from and when is it performed?
Results from the formation of base pairing, stabilised by hydrogen bonding
Performed only after template is denatured by heat
What type of process is annealing and renaturation?
Annealing and renaturation are a competitive process
Between annealing and renaturation, what occurs in preference and due to what reason?
Annealing of primer occur in preference to renaturation due to the vast excess of the primer
What does DNA polymerase require to function in PCR?
- Template strand with primer
- Deoxynucleotide triphosphates
- Mg2+
- Roughly neutral pH
What is PCR based on and what are they reliant upon and list them?
PCR based on 3 states reliant upon hybridisation of primers and formation of partial duplex:
- Denatured
- Annealed
- Native state at the optimal extension tempearture and pH for enzyme activity
What must be thermostable and due to what reason?
Polymerase must be thermostable as in order for PCR to work, the reaction must go through multiple rounds of extreme heating and cooling
What is the definition of thermostability?
The ability to retain activity upon repeated heating to temperatures that would destroy most enzymes
From what bacterium is a polymerase is used due to thermostability?
A polymerase from a thermophilic bacterium is used
Steps involved in PCR
- Template, primers, enzyme and reactants mixed
- Denaturation at 95 degrees celcius
- Anneal at the Tm of the primers
- Extend from the 3’ end of the primer at 72 degrees
- Initiation complex formed and polymerase recognises it
