PCR (described here) functions mainly because of two components – a heat-stable DNA polymerase enzyme (adds nucleotides to a new chain of nucleotides) and a pair of DNA PCR 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 suggest, prime the nucleic acid template for the attachment of the polymerase – the DNA strand-making enzyme. 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; Figure 1) because of the ‘direction’ of DNA (See DNA Structure for more background).
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.
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 base-paired nucleotides in the entire human genome.
While that all sounds very convincing, in reality, primers designed to detect viruses often share significant amounts of homology with the human genome – sometimes resulting in false-positive amplification. 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.
Next, we’ll list a few 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.
This excess drives the formation of PD. Two main concerns arise from PD formation.
- Because PD formation is so efficient, it rapidly consumes dNTPs and primers and generates amplification inhibiting pyrophosphates. All of which can prematurely plateau the exponential accumulation of product.
- If we using a dsDNA-associating fluorescent molecule to follow the PCR’s progress during real-time PCR, then PD will also show up, and, at least during the kinetic portion of the assay, cannot be differentiated from the signal of specific amplicon accumulation.
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.
Mispriming occurs because of poorly optimised conditions or because we haven’t checked whether our sequence will inadvertently bind to an entirely different target entity e.g. a region of the human genome instead of the intended virus genome. Sometimes it just happens.
Mispriming can usually be avoided by more intensive comparison of the primer’s sequence against the GenBank database using the Basic Local Alignment Search Tool (BLAST) at NCBI. Of course, a BLAST comparison will only find matches among those sequences housed in the database. When it comes to PCR where a single nucleotide mismatch can cause amplification to fail, or at least perform with reduced efficiency, BLAST’ing primers can lead to a feeling of very false security.
In some instances, the homology of the primer to its template may indicate a perfect match simply because viral variants have not yet been sequenced and submitted. Also, because there may be many undiscovered viruses and unsequenced non-viral genomes in the world, obviously none of which are represented on GenBank, a specific match, or a “no match”, does not mean that you have exhausted your search for homologues. Take it all with a grain of salt. Designing two pairs of primers around the target region is a good place to start. This helps address the unexpected.
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 another section. —work in progress—
- A crowd-sourced database of virus primers. www.virusprimers.org/
- The mechanics of the polymerase chain reaction (PCR)…a primer
- Reverse transcription-polymerase chain reaction (RT-PCR)…a primer
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- Mackay IM, Mackay JF, Nissen MD and Sloots TP. Chapter 1: ”Real-time PCR; History and fluorogenic chemistries”, In: Real-Time PCR in Microbiology, IM Mackay (Editor) 2007. Caister Academic Press, Norfolk, UK.
- Mackay IM, Bustin S, Andrade JM, Kubista M and Sloots TP. Chapter 5: ”Quantification of microorganisms: not human, not simple, not quick”, In: Real-Time PCR in Microbiology, IM Mackay (Editor). 2007. Caister Academic Press, Norfolk, UK.
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- Mackay IM. Respiratory viruses and the PCR revolution. In: PCR Revolution: Basic technologies and applications, Bustin, SA (Editor). 2010. Ch 12. Pp189-211. Cambridge University Press.
- This post from 05MAY2015 was posted over on my old blog platform virologydownunder.blogspot.com.au. It has now been moved to here.