It is important to give careful consideration to the locations and characteristics of primers, probes, and amplicons before starting any real-time PCR experiment. Particularly crucial for primers and probes is ensuring you have an appropriate melting temperature (Tm), which determines the conditions under which these will bind to your target sequence. This article provides general guidelines for designing primers and probes and choosing target locations for amplification.
When calculating melting temperatures using online tools, such as the IDT SciTools® OligoAnalyzer® Tool, be sure to enter the appropriate real-time PCR conditions from your experiment. Commonly used parameters are: 50 mM K+, 3 mM Mg2+, and 0.8 mM dNTPs; however, reaction conditions can vary widely from this, particularly with respect to Mg2+ concentration. Therefore, to obtain the Tm values for your specific experimental conditions, it is important that you use your own reaction parameters.
IDT recommends that you aim for PCR primers between 18 and 30 bases; however, the most important considerations for primer design should be their Tm value and specificity. Primers should also be free of strong secondary structures and self-complementarity. Design your PCR primers to conform to the following guidelines:
- Melting temperature (Tm): The optimal melting temperature of the primers is 60–64°C, with an ideal temperature of 62°C, which is based on typical cycling and reaction conditions and the optimum temperature for PCR enzyme function. Ideally, the melting temperatures of the 2 primers should not differ by more than 2°C in order for both primers to bind simultaneously and efficiently amplify the product.
- Annealing temperature (Ta): The annealing temperature chosen for PCR relies directly on length and composition of the primers. This temperature should be no more than 5°C below the Tm of your primers.
One consequence of having Ta too low is that one or both primers will anneal to sequences other than the intended target because internal single-base mismatches or partial annealing may be tolerated. This can lead to nonspecific PCR amplification and will consequently reduce the yield of the desired product. Conversely, if Ta is too high, reaction efficiency may be reduced because the likelihood of primer annealing is reduced significantly. Optimal annealing temperatures will result in the highest product yield with the correct amplicon.
- GC content: Design your assay so that the GC content is 35–65%, with an ideal content of 50%, which allows complexity while still maintaining a unique sequence.
Primer sequences should not contain regions of 4 or more consecutive G residues.
You have a choice of using single-quenched or double-quenched probes. IDT recommends use of double-quenched probes because they provide consistently lower background, resulting in higher signal compared to single-quenched probes. Double-quenched probes that include the IDT ZEN™ or TAO™ molecule as a secondary, internal quencher allow for longer probe lengths to be used in addition to providing strong quenching and increased signal. (Read more about ZEN and TAO quenchers in the article, Two Quenchers are Better Than One!,) If designing single-quenched probes, ensure that they are 20−30 bases in length. This will help you to achieve an ideal Tm without increasing the distance between the dye and quencher such that the quencher will no longer optimally absorb the fluorescence of the dye. Design your PCR probes to conform to the following guidelines:
- Location: Ideally, the probe should be in close proximity to the forward or reverse primer, but should not overlap with a primer-binding site on the same strand. Probes can be designed to bind to either strand of the target.
- Melting temperature (Tm): Preferably, probes should have a Tm 6–8°C higher than the primers. If the melting temperature is too low, the percentage of probe bound to target will be low. In this case, the primers may amplify a product, but sensitivity may be compromised as all target sites are not saturated with probe resulting in reduced fluorescence signal that does not truly represent the true amount of target present in the sample.
- Annealing temperature (Ta): The annealing temperature should be set no more than 5°C below the lower primer Tm. Use this as a general guideline, but note that optimization may still be necessary.
- GC content: As with primer sequences, aim for a GC content of 35−65% and avoid a G at the 5’ end to prevent quenching of the 5’ fluorophore.
General checks for both primer and probe designs
- Complementarity and secondary structure: Primer and probe designs should be screened for self-dimers, heterodimers against the 2 primers, as well as hairpins. The ΔG value of any self-dimers, hairpins, and heterodimers should be weaker (more positive) than –9.0 kcal/mole. Positive numbers indicate that the actual secondary structure shown will not form at all. Use the free, IDT, online OligoAnalyzer Tool for this purpose.
- Specificity: Run a BLAST alignment to ensure the selected primers are unique to the desired target sequence and that probe efficiency will not be reduced due to off-target interactions (this can be done directly from the OligoAnalyzer Tool).
- Length: Typically, amplicons of 70−150 bp allow for enough nucleotide sequence within which the primers and probe with adequate Tm can be designed. This length is most easily amplified using standard cycling conditions. Longer amplicons of up to 500 bases can be generated, but cycling conditions will need to be altered to account for the increased extension time.
- Location: When analyzing gene expression, it is good practice to treat your RNA samples with RNase-free DNase I before quantification. Whenever possible, design your assays to span an exon–exon junction to reduce the possibility of genomic DNA detection and amplification.