Last Updated 06-September-2013
PCR functions mainly because of two components - a thermostable DNA polymerase and a pair of DNA 'primers'. Primers are short, made to order, stretches of oligonucleotides ('oligos' - from Greek meaning scanty or few). Modern oligos can be synthesized in lengths >100nt however the behaviour of oligonucleotides longer than 20nt is different from that of shorter oligos and different calculations are employed to determine their thermodynamic characteristics.
What is a primer...?
Primers, as their name may suggests, prime the nucleic acid template for the attachment of the polymerase. This is the first step towards duplicating that template. The primer directs the polymerase to move in a 5' to 3' direction (drawn left-to-right) because of the 'direction' of DNA (See Nucleic Acid Structure for more background).
Figure 1. DNA has direction. The polynucleotide chain shown above is 'read' in a 5' to 3' direction by the polymerase. This would be from the top to the bottom or from the phosphate group to the hydroxyl group.
Primers hybridize at a temperature that is affected by their sequence, concentration, length and ionic environment. This annealing temperature is usually referred to as the TM (melting temperature) but is in fact 5–10°C below the TM. The term TM describes the temperature at which 50% of the primer–target duplexes have formed.
Figure 2. Deoxynucleotide triphosphates. Each of the five deoxynucleotides are shown, 2'-deoxycytidine-5'-triphosphate (dCTP; C9H16N3O13P3, MW=467), 2'-deoxyguanosine-5'-triphosphate (dGTP; C10H16N5O13P3, MW=507), 2'-deoxyadenosine-5'-triphosphate (dATP; C10H16N5O12P3, MW=491), 2'-deoxythymidine-5'-triphosphate (dTTP; C10H17N2O14P3, MW=482) and 2'-deoxyuridine-5'-triphosphate (dUTP; C9H15N2O14P3, MW=468).
PCR gleans its extreme specificity from the primers. At each and every position of a new primer, we have 4 nucleotides to choose from, dATP, dCTP, dGTP and dTTP. So, if we design a sequence-specific primer of 20-30nt nucleotides in length ('20-30mer'), the chance that that exact sequence will occur randomly in nature will be 1/4 x 1/4 x 1/4 etc, 20 or 30 times i.e.
That means a 1 in 1012 to 1018 chance of a 100% homologous match to an unintended target. Or to put that in perspective, there are 2.85 x 109 basepaired nucleotides in the entire human genome. While that sounds all very convincing, in reality, primers designed to detect viruses often share significant amounts of homology with the human genome - sometimes resulting in false positive amplifications. Even when the homology is far from 100%, primers may still amplify an unintended target as shown below. This most likely reflects the co-evolution of many viruses with humans during which time they have "captured" bits of our genome and "deposited" bits of their own genome. Below are some of the problems we can encounter when using the PCR.
The first problem I'll discuss is the most common and the most difficult to avoid. Depending on your requirements, it may also be the least significant.
When a small amplicon results from the extension of self-annealed primers, you get primer-dimer (PD) i.e. a dimer of one (self-annealing) or both primers resulting in a template capable of being extended by the polymerase. PD formation is highly efficient because the primers are in vast excess compared to the amount of template or even to the number of amplicon molecules at the end of the PCR.
Figure 3. Examples of how primer-dimer (PD) amplicon can be formed. Ten examples are shown of sense and antisense primer interactions resulting in an amplicon. Note that different length amplicons can be formed. The largest PD would result from the hybridization of the smallest number of nucleotides and would approach the length of the two primers added together.
Mispriming is the result of a primer binding to an unintended template resulting in amplification. The amplicon (PCR product of a single species) can sometimes be the same size as the intended product, but is usually a different size when viewed following agarose gel electrophoresis.
This problem results from the way we design our primers. I am excluding self-annealing and secondary structures from here because we will deal with them specifically in the next section.
--work in progress---