qPCR primer design for ATMPs

Where to place the primers?

The standard rule is to position the primers over a non-natural junction to prevent nonspecific binding of endogenous DNA or transcribed RNA. Suppose the introduced gene is identical to the endogenous gene. In that case, the optimal primer location is at the promoter-target junction, the junction between the gene and the post-translational enhancement element, or over an exon-exon junction to ensure that only the vector DNA is detected.

If the assay should only detect expressed transcripts, it is crucial to design it to avoid detecting endogenous transcripts. To achieve specificity, primers cover vector-specific elements that are also transcribed, such as promoters, enhancers, or regulatory elements.

If one cannot distinguish the vector-derived transcript from the vector DNA, then one must perform DNA removal before conducting RT-PCR. Additionally, including a No Reverse Transcriptase (RT-negative control) becomes necessary to account for copies of contaminating vector DNA. If the RT-negative control, the RT-PCR without enzyme detects any signal it is originating from vector DNA sequences present before the RT-PCR. When the introduced gene is codon-optimized and has a different sequence than the endogenous gene, primer and probe sequences can target regions with the least similarity to the endogenous sequence.

The primers or probe should focus on the unnatural exon-exon junction in exon-skipping therapies.

Designing primers and probes to target the vector backbone independently of the delivered transgene in early development reduces assay development efforts by employing a universal PCR assay.

The fluorescent signal

Both qPCR and dPCR rely on fluorescent dyes or probes to quantify the amplification of the target DNA. The signal is achieved with dyes that bind to double-stranded DNA (dsDNA) by inserting themselves between the two strands of the DNA double helix, such as SYBR green, or with probes carrying a fluorescent reporter. The complementary annealing of the probe provides greater target specificity compared to intercalating dyes, which bind to double-stranded DNA independently of sequence.

Typically, the probe has a quencher at the 3′ end and a fluorophore at the 5′ end. The role of the quencher is to inhibit the fluorescence emitted by the fluorophore, ensuring that fluorescence is only detected when the probe is cleaved during PCR amplification. Probes containing two quenchers, known as double-quenched probes, allow for longer probe lengths and improve assay specificity and signal-to-noise ratio. The second quencher is internal, reducing the distance between the dye and the quenchers.

Due to its high signal intensity, FAM is a common dye for singleplex assays and low-abundance targets in multiplexed PCR assays. Whatever fluorophore you choose, it is essential to ensure that the selected quencher is compatible.

Checklist for primer design

• Blast to ensure that the binding is specific.
• Validate the primers and probes using genomic DNA or total RNA from untreated host tissue.
• Test the primers and probes in the particular tissues or biofluids relevant to each species intended for use in the study.
• Aim for amplicon lengths of 60-150 bp to ensure the efficiency of the PCR amplification process.
• Ensure that the difference between the Tm of the two primers is no more than 5°C to ensure that they bind simultaneously.
• Set the probe’s annealing temperature to 5-10°C higher than the primers to saturate the target template sequences with the probe at each amplification cycle.
• Aim for 18-30 bases for the primers and 20-30 bp for single-quenched probes. Longer probes may yield a high background signal due to the increased distance between the quencher and fluorophore.
• Maintain the GC content in the primers and probes at 35-65%, ideally 50%. G and C form 3 hydrogen bonds, while A and T only have 2. Higher GC content requires higher melting temperatures.
• Avoid placing G at the 5′ end, as it can quench the fluorophore.
• GC content in the 3′ end is beneficial as it promotes stronger binding at the 3′, supporting polymerase priming.
• Avoid G and C repeats longer than three bp.
• Ensure the primers and probes are free from secondary structures and do not form hairpins.
• Test for primer dimers using a melting curve in probe-based assays, as primer dimers may be a significant issue when amplifying low-copy-number targets.

Reference: Hays, A. et. al, Recommendations for Method Development and Validation of qPCR and dPCR Assays in Support of Cell and Gene Therapy Drug Development, AAPS Journal, 2024.

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