Polymerase Chain Reaction - PCR
SP, Oct 2005
2. Nature of amplification
3. A simple protocol
4. Primers and adapters
Target - amount, length, GC-content, secondary structures, non-intended primer binding sites, etc. E.g., long targets may require the use of special 'long template' polymerases.
Template source - purity, presence of other nucleic acid species that may have primer binding sites, etc. E.g., reverse transcription products, if not treated with RNAse H, may carry inhibitory RNA molecules.
Primers - amount, specificity of primers, GC content, secondary structures, inter-primer complementarity, dgeneracy, etc. E.g., the effective primers in a degenerate 'primer' (really a mix of multiple primers) may only be a small proportion, requiring one to use more of the degenerate primer in the reaction.
DNA polymerase - robustness, buffer requirements, fidelity, 'speed', optimal temperature, proofreading activity, ability to add A-overhangs, etc. E.g., Taq but not Pfx polymerases can add an A-overhand, facilitating TA-cloning.
Purpose of experiment - pilot, quantitative, preparative, diagnostic, etc.
Thermocycling conditions - largely dictated by above aspects.
Nature of amplification
Amplification is exponential (2 -> 4 -> 8 -> 16...) only after the first few cycles. Exponentiality is lost at high cycle numbers. The limiting cycles numbers depend on the concentration of the target in the reaction.
Amplification, when possible, that uses only one primer is linear (1 -> 2 -> 3...).
A simple protocol
Volume - 50 ul final
Template - 1 ug genomic DNA or 1 ng plasmid DNA or a tenth of a typical reverse transcription reaction
Primers - 10-20 pmoles each
dNTPs - 1 ul of a mix containing 10 mM each dNTP
Polymerase, buffer, divalent cations - follow polymerase supplier's guidelines
Extra -PCR enhancers such as DMSO, betaine, PCRX™, may be added if trying different conditions or when suspecting a difficult target
Set up the reactions on ice, adding polymerase in the end. Mix well, spinning down if needed to collect droplets, and set up thermocycling with these parameters:
Td x td - denaturation - 94°C x 30 sec
Ta x ta- annealing - 55-60°C - depending on primer sequence - x 30 sec
Te x te- extension - 68-72°C - depending on polymerase - x n - depending on polymerase - sec
Initial denaturation phase - just denaturation for 2 min
Amplification phase - cycling with above parameters
Final extension phase - just extension for 10 min
Primers and adapters
The figure illustrates below how oligonucleotide primers get 'incorporated' into the final PCR product sequence. 'Other' nucleotides in the product are incorporated by the DNA polymerase. Thus, one can introduce mutations into and add 'adapters' to the final product.