PCR and its role in Diagnostics Flashcards
What is Polymerase Chain Reaction (PCR)?
Polymerase Chain Reaction is an enzyme-based method to specifically amplify segments of DNA using a thermal DNA polymerase in a cyclic process.
What is a chain reaction?
A chain reaction is a series of events, each one of which is dependant upon the preceding event to sustain itself.
Typically it is a series of reactions that lead to an exponential increase in the number of events occurring in a sequence.
Under which conditions is PCR specific?
PCR is a method to specifically amplify segments of DNA.
It is specific only if annealing is undertaken at the melting temperature (Tm) of the primers (ie. high stringency conditions). This prevents mismatched based pairing.
How is specificity determined in PCR?
The segment amplified in PCR is determined by the sequence at the ends of the segment to be amplified; exponential amplification requires two primers corresponding to these sequences.
Specificity is thus determined by the uniqueness of these sequences and their complementarity.
What is a DNA-dependant DNA Polymerase, and what are its functions?
It is the enzyme used in PCR that 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 (ie. adds nucleotides to the 3’ end of the priming strand).
What is annealing, and how does it play a role in PCR?
Annealing is a hybridisation process where we hybridise a short, single-stranded DNA molecule to another molecule that has been denatured.
As a consequence, we’re forming a partially double-stranded molecule that can then act as a template for PCR.
Annealing results from the formation of base-pairing, stabilised by hydrogen bonding. It is performed only after the first template is denatured by heat.
Describe how we get more annealing in the reaction?
The template at the start of the reaction is in a low concentration. The formation of the primer template duplex is forced to occur by providing a huge excess of the primer.
This is in a competition between the renaturation of the double-stranded template and the annealing of the primer to the template, in which the annealing is in preference.
What does it mean when a DNA-dependant DNA Polymerase is used?
- it synthesises a new nucleic acid strand by copying a DNA molecule
- it cannot copy RNA nor make RNA
- RNA must first be copied to DNA by reverse transcription before it can be amplified by PCR
The reaction is based on transitions between three states reliant upon hybridisation of primers and formation of a partial duplex.
What are the three states?
- a denatured state (where the template becomes single stranded)
- an annealed state(the formation of a duplex with the primer and template strand)
- a native state at the optimal extension temperature and pH for enzyme activity
Why is thermostability important in PCR?
For PCR to work, the reaction must go through multiple rounds of extreme heating and cooling, so the polymerase must be thermostable.
Thermostability means ‘able to retain activity’ upon repeated heating to temperatures that would “destroy” most enzymes.
Hence, a polymerase from a thermophilic bacterium such as Thermus Aquaticus is often used (Taq Polymerase).
Briefly, what are the three steps of PCR?
We mix everything (template strand, primers, enzyme, and reactants) in a test tube.
1) we denature them at 95°C
2) we then anneal at the Tm of the primers at 55°C
3) extend from the 3’ end of the primers at 72°C
Explain how PCR is qualitative and not quantitative.
The end of the PCR reaction does not have a quantitative output and cannot be used to inform template copy number.
Regardless of the starting concentration of template, the same endpoint is reached as amplification becomes rate limited.
Thus PCR is qualitative in the sense that it can determine the presence or absence of a substance.
We use modifications to provide a measurable output for PCR.
Thus, explain how PCR is made quantitative.
There are a number of different quantitative PCR detection methods used for diagnostics. Collectively, they are referred to as ‘real-time PCR’ or ‘quantitative PCR’.
These techniques utilise fluorescent detection of the amplification. They are used for quantifying the amount of a target DNA molecule in the sample.
How is PCR used in SNP detection?
PCR can be used to detect SNPs (single nucleotide polymorphisms); several methodologies allow this, and two of them are adaptations of quantitative real-time PCR.
They rely on differences in the Tm of a duplex containing a single nucleotide mismatch.
COMMON APPLICATIONS:
- antibiotic resistance testing: TB and many other organisms
- identification of genetic markers
What are the two approaches to SNP detection, specifically?
- high resolution melting (HRM): the Tm of the amplified product is used to determine which sequence is present
- probe based version of qPCR (sometimes referred to as Allelic discrimination): where specific binding of the probe to the amplified region containing the SNP is detected
