PCR And Its Role In Diagnostics Flashcards
(39 cards)
What is meant by the term “Polymerase Chain Reaction”?
An enzyme based method to specifically amplify segments of DNA using a thermal DNA polymerase in a cyclical process
What is a chain reaction?
A series of events each of which is dependent upon the preceding event to sustain itself. Leads to an exponential increase in number of events.
Why is PCR so specific?
- Is only specific if annealing is undertaken at the melting temperature Tm of the primers
- This prevents mis-matched based pairing
What is the segment amplified determined by?
Determined by the sequence at the end
How would you amplify the segment bounded by the known sequence?
By choosing primers complementary to these ends. Exponential amplification requires two primers each complementary
What is the DNA dependent DNA polymerase enzyme used for?
- This enzyme recognises a specific structure consisting of a partially double stranded DNA forming an initiation complex with it
- The reaction extends a partially double stranded molecule from the 3’ end of the non template strand
How is a partially double stranded structure formed in PCR?
By annealing a short single stranded DNA molecule (a primer)
How is this achieved?
- The double stranded template first has to be denatured and converted into a single stranded molecule
- Performed only after the template is denatured by heat
- The newly formed strand is sometimes referred to as the nascent strand
What is annealing of the primer to the template?
Annealing is an alternative way of describing hybridisation
How is annealing of the primer achieved?
Achieved using the predicted melting temperature of the primer template duplex under high stringency conditions
What does annealing result from?
Annealing results from the formation of base pairing, stabilised by hydrogen bonding
What type of process is annealing of the primers vs renaturation?
- A competitive process
- Annealing of the primer occurs in preference to renaturation and is driven by favourable kinetics as a result of the vast excess of the primer present in the reaction
What are some basic rules about PCR?
- Enzyme used is a DNA dependent DNA polymerase
- Synthesises a new nucleic acid strand by copying a DNA molecule
- It can’t copy nor make RNA
- RNA must be first converted to DNA by reverse transcription before it can be amplified by PCR
What does the PCR reaction require?
- A template with opposing primers
- Deoxy nucleotide triphosphates
- Mg2+ ions
- A roughly neutral pH
What is the PCR reaction based on?
The reaction is based on a transition between 3 states reliant upon hybridisation of primers and formation of a partial duplex
- Denatured (template becomes single stranded)
- Annealed (formation of initiating template)
- Native state at the optimal extension temperature and pH for enzyme activity
What must be required for the PCR reaction to work?
- The reaction must go through multiple rounds of extreme heating and cooling
- So the polymerases MUST be thermostable (able to retain activity)
What are the results expected from a PCR?
Every cycle results in a doubling of the amount of product therefore is an exponential accumulation of product
- The reaction has characteristic kinetics determined by depletion of reaction and the acidification of the reaction
What are diagnostic tools used for?
Used for identification, Confirmation and Quantification of specific DNA sequence
Give some examples where diagnostic tools are used?
- Presence absence calling TB - detection in sputum, determining treatment choice
- Differentiating between closely related organisms “Swine flu vs human influenza” both H1N1
Why can’t the PCR reaction inform the template copy number?
The end of the PCR reaction does not have a quantitative output and can’t be used to inform template copy number
What is the cause for this case?
- Same end point as amplification becomes rate limited
- This is independent of the starting concentration of template
How is this problem solved?
We therefore use modifications of this technique to provide measurable output during the exponential phase of the amplification in real time
What is quantitative PCR methods also known as?
There are a number of different quantitative methods which are collectively referred to as real time PCR or quantitative PCR
What do these Real time PCR techniques utilise?
- These techniques utilise fluorescent detection of the amplification
- Are used for quantifying the amount of a target DNA molecule in the sample
