The design of quantitative PCR (qPCR) assays has come a long way since the melting temperature of each oligo was estimated by eye. Today, scientists can reference sequences from a multitude of publications, select from a database or catalogue of predesigned assays or even purchase inventoried oligos with qualified performance characteristics. But there are still occasions when scientists want to target a different region entirely, distinguish between closely related sequences or design oligos to a completely novel target. Free design software programs are readily available to expedite this process and eliminate the guesswork associated with manual design, but insight from the researcher remains essential to ensure successful amplification.
The most common issues with de novo assay design relate to the software failing to propose a candidate assay, or else an assay design that does not perform to expectations. Often, repetitive elements, nonspecific amplification or adenine-thymine rich (AT-rich) regions with low melting temperatures (TM) are the culprits. In the case of poor assay performance, a scientist may observe signal generation that is little to none, unexpected cycles-to-threshold (Cq) values or irreproducible results. How can these impediments be avoided?
Here are a few tips for avoiding or overcoming common qPCR assay-design issues:
Tip 1: Know what to expect. Targets represented at a low concentration are much harder to detect, such as a rare mutant allele in a background of wild-type. The inherent variability from pipetting only one or a few molecules will produce stochastic results. Further, each assay will have a limit of sensitivity beneath which a target sequence cannot be reliably detected. This threshold depends intimately on probe chemistry, reagent quality and enzyme efficiency. If the limit of sensitivity is not properly characterized, then interpretation of data is compromised and becomes more qualitative than quantitative. To verify that the target of interest is present in the samples under investigation, consider applying alternative techniques such as RNA SEQ or single-molecule FISH.
Tip 2: Be specific by design. Software is only as good as its input, and it cannot account for all the biological considerations of an investigation. Should the assay span an exon-exon junction to better analyze gene expression? Should it detect all isoforms or only a subset with alternative splicing? Are there homologous sequences that must be distinguished or else tolerated? Additional bioinformatic analysis will help avoid off-target amplification, but selecting the appropriate genetic method is also important to ensure a proper outcome: fluorogenic probes or DNA-binding dyes? Allele-specific probes or primers? Software algorithms cannot control for all contingencies, such as unanticipated variants, so a baseline failure rate is unavoidable with de novo assay design.
Tip 3: Select dyes to suit the real-time thermal cycler. Reporters that fluoresce at a longer wavelength than FAM should be carefully matched to the instrument optics. These fluorophores typically are used for multiplexing when it becomes critical to resolve overlapping signals. Single-wavelength excitation sources and narrow band-pass filters restrict the dyes that can be accommodated, and subtle differences in spectra interfere with the deconvolution algorithms. Consult instrument documentation and dye recommendations, especially because pH and temperature dependencies can reduce dye intensity. Fluorophores designed for microarrays or protein labeling may not work well in qPCR.
The normalized emission spectra for a series of fluorophores provide a reference to choose potential candidates for qPCR.
Optimal reporters for a pentaplexed assay are identified by comparing spectra to the instrument’s filter specifications.
Tip 4: Select your polymerase or master mix according to the application. Examples of applications include multiplexed amplification of several targets in unison, mismatch discrimination for allelic discrimination and high-fidelity PCR for subsequent manipulation. If your target sequence is a bacterial gene, be aware that many polymerase preparations have contaminating bacterial DNA that obscures detection of dilute target samples.
Tip 5: Qualify new assay designs using positive controls. Regardless of how well the assay is designed, it may not yield the expected result. Use appropriate controls, either carefully-quantified plasmids or a cell line with known gene expression, to verify the quality of the nucleic acid following extraction and the performance characteristics of the assay. When designed appropriately, two different assays that target the same gene should produce similar Cq values. Redundancy will bolster confidence in the results.
Tip 6: Guidelines or myth? Much of the PCR process remains a mystery, particularly during the earliest cycles of amplification. Assay design was built upon tribal knowledge, with some recommendations questionable and others inviolable. The rule that a probe must bind at a temperature 10˚C higher than the primers was developed with dated thermodynamics. More accurate predictions may be made using SantaLucia’s unified nearest neighbor values, which suggest a more modest difference between primers and probe. Although guidelines improve the probability of success, many rules can be broken without consequence—if accompanied by an appreciation for experimentation.
Christina Ferrell, Ph.D. is a Technical Applications Specialist at Biosearch Technologies. She has over 20 years of experience in Life Sciences and has published several peer-reviewed articles involving quantitative real-time PCR. Christina presents other helpful tips for qPCR in the BiosearchTech “Referral from the Doctor” Blog series.