PCR 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.
A chain reaction
- A Chain reaction is a series of events each one of which is dependent 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
Complementarity of the primers
- Is specific only if annealing is undertaken at the melting temperature Tm of the primers, ie high stringency conditions
- This prevents mis-matched based pairing
- Since the segment amplified is determined by the sequence at the ends
- If we want to amplify a segment bounded by known sequence we can do this by choosing primers complementary to these ends
- and exponential amplification requires two primers each complementary
DNA dependant DNA polymerase
• The enzyme recognises a specific structure consisting of a partially double stranded DNA forming an initiation complex with it.
Primer/ template duplex
- In PCR a partially double stranded structure is formed by annealing a short single stranded DNA molecule (a primer)
- In order to achieve this, the double stranded template has first to be denatured and thus made into single stranded molecule
- It is performed only after the template is denatured by heat
- The newly formed strand is sometimes referred to as the nascent strand
Annealing of the primer to the template
- Annealing is an alternative way of describing hybridisation
- Annealing of the primer under high stringency conditions is achieved using the predicted melting temperature of the primer-template duplex
- Annealing results from the formation of base-pairing, stabilised by hydrogen bonding
Annealing & Renaturation
Competitve processes
• 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
In PCR the enzyme used is DNA dependant 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 converted to DNA by reverse transcription before it can be amplified by PCR
The reaction requires (for DNA polymerase)
- A template strand with a primer (usually 20-30 bases long) annealed to it
- Deoxy nucleotide triphosphates (dATP, dGTP, dCTP, dTTP)
- Mg2+ ions
- A roughly neutral pH
PCR application
Diagnostics - routine diagnostic tool used for identification, confirmation and quantification of specific DNA sequence
Examples of PCR Diagnostics
- Presence absence calling TB - detection in sputum, determining treatment choice
- differentiating between closely related organisms “swine flu vs human influenza” both H1N1 subtypes
- How much is present - determining when treatment might be commenced, “HIV viral load”
- Identifying individuals positive for SARS-CoV-2 and thus have COVID-19
The end of PCR reaction
does not have a quantitative output and cannot be used to inform template copy number
PCR and quantitive analysis
- 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 utilize fluorescent detection of the amplification
- Are used for quantifying the amount of a target DNA molecule in the sample
What does SNP stand for?
Single nucleotide polymorphism
Common application of SNPs
- Antibiotic resistance testing -TB and many other organisms
- Identification of genetic markers - drug sensitivity/catabolism (CYP2C9 and VKORC1 variants confer warfarin sensitivity),markers of disease (Cancer) or treatment response (HCV),
