Abstract
PCR Perfection: insider tricks was a 1-day symposium on the actual design methods of PCR assays. This day primarily focused on different design methodologies for ensuring an optimal PCR reaction.
For many years now, real-time PCR has been a staple technique in research, diagnostics and development. With this, many new and important discoveries have been made in the fields of diagnostic sciences, such as the detection of possible cancer-causing gene mutations through SNP-analysis . Real-time PCR has enabled the identification of possibly fatal infections in hours, instead of days. This is particularly true in meningitis/encephalitis cases where early detection of the relevant pathogenic organism aids physicians in ensuring the correct treatment is administered.
It has long been debated how best to design a real-time PCR assay or indeed any type of PCR assay. It is common to find differences in design and performance methodologies between laboratories. I have heard the debate regarding where best to place primers and whether to use probes or agarose gels in the detection of PCR products at various courses. Usually with these courses, the focus is on the end product and the data generated, not the actual methods of how the assay was designed. This is usually spoken about later, one-to-one, rather than in a grander forum. So it was very refreshing to find a symposium where the focus was on the actual PCR assay design methods and the range and lengths that different researchers and groups will use when designing an assay. The course also, but to a lesser extent, had a small debate discussing which detection method was preferred, but the central theme remained the actual design and optimization of the PCR assay.
PCR assay design: the importance of getting the fundamentals right
The core of the meeting comprised three talks, each tackling the issue of good assay design. Each speaker had their own way of ensuring that their PCR assay was optimal and had their own method of doing so.
First to speak was Rob Powell of Primer Design Ltd. Through his work as a research fellow and now as an assay designer, he demonstrated how secondary structure in amplicons can affect primer-binding efficiency and amplification ability. The formation of a secondary structure at any point in the primer-binding site can cause the primer to either not prime or not to prime efficiently. Thus, it is important to ensure that secondary structures either do not exist in your amplicons or that they do not form at the primer annealing and extension temperatures. Through his research as a fellow at the University of Southampton, UK, Powell provided published examples of how secondary structures in an amplicons had led to poor melting-curve analysis in Sybr green assays. This resulted in the data being neither accurate nor reproducible.
The second speaker to address this subject was David Sugden of Kings College, London, UK. Sugden took a different approach to PCR assay design and reaction optimization and advocated using universal reaction conditions throughout his work. That is the same primer concentration for all his assays (500 nM per reaction); the same primer annealing temperature and template input. His methods did agree strongly with Powell’s at the beginning, when he also put emphasis on good primer design and placement being paramount in assay design. Sugden also explained his use of the Universal Probe library available free at Roche online as part of their off-the-shelf assay offer. The primer design software required only that the gene sequence of interest be input; the software would design the several sets of primers, of which one could then select oneself. But it is necessary for the user to ensure that the primers are specific to one’s target. The aim of the software being that one then buys these primers together with the 8-base probe supplied by Roche for that primer set. Though this is optional, the software can be used just for primer designing purposes.
The third speaker in this trio was Stephen Bustin of Queen Mary’s College, University of London, UK. Assay design was pursued from a different angle. Again, good primer design was fundamental to assay design. In contrast to both the previous speakers, it was suggested that multiple sets of primers should be designed per amplicons. This, coupled with full-assay optimization (in stark contrast to Sugden), should be carried out to ensure maximum amplification efficiency. Other important factors from sample and nucleic acid integrity were also discussed.
A very important point was raised and presented by Stephen Bustin. He discussed the importance of reading and interpreting results correctly. The publication of incorrect and misleading information in real-time PCR-assay design and data produced from these assays has led to the public being given misleading information. In one instance (in which Bustin was an expert witness), data regarding measles and autism were published indicating that measles was detected in all cases of autistic children. On closer inspection of the data, nonexponential rises in Taqman® florescence was reported as positive when it was clearly not. Other examples included the publication of incorrect primer sequences, some of which were not even on the same gene that was being reported.
Conclusion to the day
This day highlighted the key differences in the way people design assays. It is impossible to say which method is the right method but it did indicate that there is not a one-size-fits-all method.
The core message of the day was: when designing an assay, get your primers right. Ensure that specificity is high and that variable regions are avoided. From here, it is up to the designer as to how they proceed. It is possible to go in many different directions: a universal design approach as presented by Sugden, an assurance of amplification efficient advocated by Powell or additional assurance of primer specificity and sensitivity through multiple primer sets from Bustin. It is up to the individual to decide.
Personally, I found this day to be very interesting; in particular, what I have written about here. It has brought to light the different methods I can bring to my own work to ensure that PCR-assay design is to the highest quality that it can be.
Financial & competing interests disclosure
William Rivenberg has, in the past, bought and used kits supplied by Primer Design Ltd and has a working relationship with Rob Powell. The author has no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of this manuscript.