How to design primers and probes for PCR and qPCR

It is important to give careful consideration to the locations and characteristics of primers, probes, and amplicons before starting any PCR or qPCR (quantitative PCR, also known as real-time PCR) experiment. Particularly crucial for primer and probe design is ensuring you have an appropriate melting temperature (T_m), which determines the conditions under which these oligos 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 and PrimerQuest™ Tool, be sure to enter the appropriate conditions from your PCR or qPCR 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 T_m values for your specific experimental conditions, it is important that you use your own reaction parameters.

How to design PCR primers

For PCR primer design, IDT recommends that you aim for PCR primers between 18 and 30 bases; however, the most important considerations for primer design should be the T_m value and on-target binding efficiency. Primers should also be free of strong secondary structures and self-complementarity. Design your PCR primers according to the following guidelines suggested by IDT scientists:

• Melting temperature (T_m): 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 (T_a): The annealing temperature chosen for PCR relies directly on T_m of the primers. This temperature should be no more than 5°C below the T_m of your primers. One consequence of having T_a 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 T_a is higher than the T_m of the primers, 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 of your oligonucleotides 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.

qPCR probe design

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, Double-quenched probes increase signal to noise ratios by decreasing background fluorescence. If designing single-quenched probes, ensure that they are 20–30 bases in length. This will help you achieve a suitable T_m 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 (T_m): Preferably, probes should have a T_m 5–10°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 quantitative accuracy may be compromised as all target sites are not saturated with probe resulting in reduced fluorescence signal that does not represent the true amount of target present in the sample.
• Annealing temperature (T_a): The annealing temperature should be set no more than 5°C below the lower primer T_m. 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.

Primer and probe design considerations

• Complementarity and secondary structure: Primer and probe designs should be screened for self-dimers, heterodimers against the 2 primers, and hairpins. The ΔG value of any self-dimers, hairpins, and heterodimers should be weaker (more positive) than –9.0 kcal/mol. Positive numbers indicate that the actual secondary structure shown will not form at all. Use our free online OligoAnalyzer Tool for this purpose.
• On-target binding efficiency: Run a NCBI 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).
• Amplicon length: Typically, amplicons of 70–150 bp allow for enough nucleotide sequence within which the primers and probe with adequate T_m 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.
• Amplicon location: When analyzing gene expression, it is good practice to treat your RNA samples with RNase-free DNase I to remove any residual genomic DNA (gDNA) before quantification. Whenever possible, design your assays to span an exon-exon junction to reduce the possibility of gDNA detection and amplification.

Free PCR and qPCR design tools!

IDT offers several free online tools (SciTools Web Tools) for oligonucleotide design and analysis. These tools contain design engines that use sophisticated formulas that, for example, take into account nearest neighbor analysis to calculate T_m, and allow you to begin your project sooner by easily analyzing and troubleshooting experimental designs.

• PrimerQuest Tool—use to generate highly customized designs for qPCR assays and PCR primers. 
• PrimeTime™ qPCR Assay Selection Tool—use to select predesigned sequences for human, mouse, and rat targets.
• RealTime qPCR Design Tool—use to design primers, probes, and assays across exon boundaries for gene targets in species other than human, mouse, and rat. 
• OligoAnalyzer Tool—use to analyze oligonucleotide melting temperature, hairpins, dimers, and mismatches. BLAST analysis can also be done directly from this tool.
• UNAFold Tool—use to analyze oligonucleotide secondary structure.

For an overview of our predesigned qPCR oligonucleotides and the basics of designing customized PCR primers and hydrolysis probes with the PrimerQuest Tool, see the article Primer design tools for PCR & qPCR.

RUO23-1699_001.1