3. PCR and Its Role in Diagnostics Flashcards
What is PCR?
Polymerase Chain Reaction is 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 Chain reaction is a series of events each one of which is dependant upon the preceding event to sustain itself.
- leads to an exponential increase in number of events.
How is specificity sustained?
Only if annealing is undertaken at the melting temperature Tm of the primers, ie high stringency conditions. This prevents mis-matched based pairing.
How is the segment amplified determined?
The segment amplified is determined by the sequence at the ends and exponential amplification requires two primers. If we want to amplify a segment bounded by known sequence we can do this by using primers complementary to these ends.
How does DNA polymerase work?
• Complementary copy of the opposing template strand is synthesised.
• The 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 (template annealed with short strand of ssDNA) from the 3’ end of the non-template strand.
- (Partially dsDNA is formed after annealing ss primer to a ss denatured template - stabilised by H bonds b/w bases).
How is annealing favoured over renaturation?
The annealing & renaturation are a competitive processes
- Template: low copy number
- Primer: very high copy number
Rules of DNA polymerase
- 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 cDNA by reverse transcription before it can be amplified by PCR
PCR transitions between which 3 states?
• Denatured (template becomes single stranded)
• Annealed (formation of initiating template)
• Native state at the optimal extension temperature and pH for enzyme activity (for elongation)
* The transition depends upon hybridisation or primers and formation of a partial duplex.
Requirement of DNA polymerase
1) A template strand with a primer (usually 20-30 bases long) annealed to it
2) Deoxy nucleotide triphosphates (dATP, dGTP, dCTP, dTTP)
3) Mg2+ ions
4) Enzyme needs to be thermostable - retain activity with repeated heat applied. ~ polymerase from a thermophilic bacterium often used (Taq polymerase).
Describe events of typical PCR cycle
1) Template, Primers, Enzyme & Reactants are mixed together
2) The template is denatured with high temperature.
3) Primers anneal with DNA at Tm of the primers.
4) The partially dsDNA extends from the 3’ end of the primer
5) The reaction is reheated and cooled again. - Cycling typically 30-40 times.
Describe the kinetics of the reaction
- The reaction has characteristic kinetics determined by depletion of reactants and the acidification of the reaction.
- Sigmoidal shape of the curve because as the reaction progresses, the conditions are becoming more acidic. – consequences of the addition of the dNTPs to the elongating strand, and also producing pyrophosphate in the elongating process
Applications of PCR
1) Diagnostics
2) Diagnostics
3) SNP detection
4) Forensics and law enforcement ~ Amplification of genetic markers
5) Amplification prior to NGS and cloning or sequencing
6) Manipulating and modifying DNA
7) Important in recombinant DNA technology
3) Amplification of genetic markers ~in forensics and law enforcement: - parentage/kinship, identification, matching sources at crime scene, authentification of biology material (cell lines, food purity).
4) Amplification of material before NGS - e.g. Simultaneously sequencing large number multiple PCR products of candidate Cancer genes.
5) Amplification prior to isolating individual segments of DNA before cloning of sequencing
6) Manipulating and modifying DNA - e.g. introducing mutations or restriction sites.
7) Important in recombinant DNA technology - for vaccines and pharmaceuticals (interferons, clotting factor, tissue plasminogen activator etc).
How is PCR used in diagnostics?
Used for identification, confirmation and quantification (qPCR used) of specific DNA sequence.
Examples:
• Presence absence calling TB - detection in sputum, determining treatment response/drug efficacy
• Differentiating between closely related organisms “swine flu vs human influenza” both H1N1 subtypes
• How much: determine when treatment might be commenced, “HIV viral load”
What is qPCR and why is it used? and how?
Real-time PCR or quantitative PCR
As there is the same end point of PCR, as amplification becomes rate limited - so it doesn’t reflect the amount of template in the mixture.
To detect amount of target DNA used, real-time PCR (or quantitative PCR) is used (modification of PCR) ~ this is includes the use of fluorescent detection of the amplification.
- Serial dilution of the template produces curves shifted to the right (fluroescence detected at higher cycles).
- The crossing point of the amplification is determined and is proportional to the template conc. at the start.
How is PCR used for SNP detection?
SNPs are used as genetic markers for association studies.
* Two methodologies, (HRM and probe based version of qPCR), which are adaptations of quantitative real-time PCR. ~ They both depend on differences Tm conferred upon short sequences of DNA by their nucleotide composition ~ of a duplex containing a single nucleotide mismatch
- HIGH RESOLUTION MELTING (HRM)
- PROBE BASED VERSION OF qPCR (aka Allelic discrimination)
