PCR assay design, simplified: qPCR, SNP genotyping and gene expression

Design is the foundation for PCR success. Get the primers and probes right, and you’ll be set up for on-target amplification, reliable Cq values and confident calls. Below is a practical run-through of the best practices for designing primers and probes for qPCR, SNP genotyping and gene expression, as well as how design software can simplify the process.

Core primer design rules

Without well-behaved, specific primers, your PCR will never perform to the required standard of sensitivity and specificity. However, there are a lot of factors that will influence primer behaviour. The following criteria can be followed for hydrolysis probe-based qPCR, SNP genotyping and gene expression assays: 

• Primer length and T_m: Approximately 18–24 nucleotides long, with melting temperatures
  typically 55–60 °C and within 1–2 °C of each other. For PCR with intercalating dye (e.g. SYBR Green), a primer T_m of 60–65 °C is preferable for increased specificity. 

• GC content: Keep within 50–60%. Avoid GC runs of three or more bases. Place a G or C at the 3ʹ end, if possible. In the last five 3ʹ bases, have no more than two consecutive Gs and Cs, and a maximum of three Gs and Cs in total. 

• Amplicon length (qPCR): Aim for 75–150 bases for efficiency and sensitivity. Shorter amplicons amplify more efficiently and are better for quantitative analysis. 

• Specificity: Check that the primer sequences are unique to your target. Run BLAST searches against the relevant genome to verify primers don’t significantly match off-target sequences, such as paralogues and repeats (unless this is specific to an organism you wish to detect). 

• Secondary structures and dimers: Analyse primers for self-complementarity and dimers. Primers shouldn’t form strong hairpin loops or primer-dimers with themselves or with each other. Online tools can identify hairpins and dimers, allowing you to adjust sequences if needed. 

• Efficiency: Check the efficiency of your primers by doing a standard curve with serial dilutions of template. Efficiency should be 90–110%, meaning the PCR doubles the template each cycle in the exponential phase. 
   

Hydrolysis probe qPCR 

In hydrolysis probe-based qPCR, a dual-labelled oligonucleotide probe binds to the amplicon and yields fluorescence upon cleavage by polymerase. A well-behaved probe is just as important as good primers, since it’s required for generating a clear signal. 

• Probe location: Place the 5ʹ end of the probe close to the 3ʹ end of the hydrolysing primer (within a few dozen bases) but not overlapping the primer’s binding site. This allows the probe to bind and be cleaved efficiently without hindering primer annealing or extension. 

• T_m: 5–10 °C higher than the primers’ T_m so it hybridises to the template before the primer and ensures efficient cleavage. 
  
  
• Length and composition: Probes are typically around 20–25 bases long, unless using modifications such as MGB and LNA. As with primers, aim for a balanced GC content (40–60%). Avoid 5ʹ G as it can quench some dyes.
  
  
• Quenchers: BHQ^® Probes are a workhorse choice thanks to low background; double-quenched BHQnova^® format helps when probes must be longer or additional sensitivity is needed. 
  

• Specificity and mismatch avoidance: The probe should ideally cover a region without common polymorphisms (unless it’s for SNP genotyping, see below) and with minimal secondary structure in the target. If polymorphisms are unavoidable, long probes can be more tolerant to mismatches when detecting targets with known variations. Verify that the probe sequence is unique to the target sequence using BLAST. 

Designing probes can be challenging, but following these criteria will help your probe to bind efficiently and produce a clear fluorescent signal when the target is amplified. 

SNP genotyping by end-point PCR 

SNP genotyping via PCR often uses a specialised form of probe design to discriminate alleles. A common approach is the allele-specific hydrolysis probe assay, where two different probes detect the two SNP variants in one reaction. The primers for SNP genotyping are usually not allele-specific; they flank the SNP site and amplify the region containing the SNP. 

Key points for SNP genotyping probe design: 

• Probe strategy: Short probes with the SNP near the centre maximise mismatch discrimination. 

• Dyes: Pick spectrally distinct reporters supported by your instrument (e.g. FAM and CIV). 
• MGB or BHQplus^® probes: Duplex-stabilising chemistry raises the melting temperature of short probes, allowing you to achieve greater single-base mismatch sensitivity.  

In practice, a well-designed SNP PCR assay will produce a distinct fluorescence end-point for each allele probe. Careful probe design will help to significantly reduce binding with a single base mismatch, however some cross-talk will remain. Endpoint cluster plot analysis will help to make distinct genotyping calls. Software can assist in designing allele-specific probe sets, taking into account the need for balanced T_m and minimising cross-reactivity. 

Gene expression (RT-qPCR) assay design 

Many of the standard primer/probe design rules still apply to RT-qPCR assays. However, there are a few additional considerations to ensure accurate measurement of mRNA levels:  

• Exon-exon junction targeting: Designing primers or probes spanning an exon-exon junction of the gene transcript greatly reduces the risk of signal from contaminating genomic DNA in your RNA sample. 

• Amplicon: Aim for a short amplicon (80–120 bp) for more efficient amplification, avoiding strong structure/repeats. 

• Isoforms: Decide up-front if you are targeting shared exons across multiple isoforms or a unique exon for a specific isoform. 

• Reference genes: Gene expression studies usually involve measuring a housekeeping gene for normalisation. You could design a separate primer/probe set for a stable reference gene (like RPLP0) and run it in a duplex reaction with your target gene. If so, ensure the two assays are compatible and that the primers don’t compete or form dimers. 

• RT primer: Take extra care designing the primer that will be responsible for the RT step. The rest of your reaction depends on the successful reverse transcription.  
  

Multiplex qPCR 

Multiplex qPCR refers to running two or more assays in the same reaction tube to simultaneously detect multiple targets. This approach is powerful as it produces more data per run and can include internal controls in the same well. However, this additional complexity requires extra care in assay design to ensure all targets amplify efficiently together.  

When designing multiplex PCR assays, consider the following best practices: 

• Uniform primer conditions: All primer pairs in a multiplex must work under the same PCR conditions. Design all primers to have very similar T_m values and ideally target amplicons of comparable length.

• Avoid cross-reactivity: Check all primers for significant complementarity, especially at the 3’ ends, because of the risk of primer-dimer formation. Ensure that each primer pair is specific for its intended target and doesn’t amplify the other targets.  

• Distinct fluorophores: For each target that you need to distinguish between, select one dyewith minimal spectral overlap and proper calibrations. Use compatible quenchers for each probe (BHQ is widely supported). 

• Optimisation and validation: Even with careful design, multiplex assays typically require some optimisation. Test each primer/probe set in a singleplex reaction to confirm efficiency and specificity. Then, combine them stepwise and test the multiplex. Adjust primer or probe concentrations so that all targets amplify with similar efficiencies. A successful multiplex will reliably deliver the required sensitivity and specificity for your application. 

Designing multiplex assays manually can be very complex – keeping track of multiple primer interactions and fluorescence spectra is a lot to juggle. This is where software design tools become especially helpful, for example automatically checking all primers in the pool are compatible.  

An all-in-one free software solution 

Designing primers and probes by hand, is time-consuming and difficult. Software can take the heavy lifting and achieve best practice each time: correct T_m models, dimer screening, genome checks, exon-junction logic, dye/quencher compatibility and multiplex harmonisation.  

Some popular options include Primer3, NCBI Primer-BLAST and commercial or supplier-provided tools. They significantly speed up design and often integrate multiple checks in one package. 

PCR Forge is the newest entry into this arena. This free, web-based software is tailored for modern assay design. 

PCR Forge is: 

• The only platform that specifically designs BHQ, BHQplus and BHQnova probes, 

• Capable of designing true multiplex assays – harmonising T_m, amplicon lengths and checking inter-assay dimers – for up to five PCR/qPCR assays or two genotyping assays 

• User-friendly and free to use for anyone to adopt best-practice assay design

• Designed to process up to 100 sequences in batch mode, 

• Able to specifically design primers for exon junctions, 

• Customisable with up to 80 design parameters and choice of three default settings for strict, moderate and relaxed designs to balance potential designs and sensitivity requirements. 
  

The right software can help avoid costly errors and save days of iteration. Try PCR Forge to generate BHQ-optimised singleplex or multiplex assays in minutes and move straight to testing with confidence.