Primer and Probe Design

This chapter focuses on the basics of ddPCR and the tools available to help you design your very own ddPCR assay.

If you’d like to create a simple gene-target detection assay, you’ll need:

  • Primer pair sequences

  • (Optional) Probe sequence

For singleplex detection (one target at a time), you can use a DNA intercalating dye like EvaGreen, where probes aren’t necessary, and primers alone suffice. However, for multiplexing, it’s recommended to develop primer-probe pairs from the outset.

Tools for Primer and Probe Design

The most well-known online tool for primer sequence development is Primer3Plus. Paste your target locus sequence, set optimal amplicon sizes (60–200 bp; the shorter the amplicon - the better the reaction efficiency), and ensure:

  • Primer Tm ≤ 65°C.

  • GC content: 40–60%.

  • Avoid excessive G/C repeats.

Recommended Primer3Plus Settings:

General Settings:

  • “Concentration of divalent cations” = 3.8 mM.

  • “Concentration of dNTPs” = 0.8 mM.

  • “Mispriming/Repeat Library” = your organism of interest.

Advanced Settings:

  • “Thermodynamic parameters” and “Salt correction formula” = SantaLucia 1998.

To start designing primers, you’ll need the gene locus sequence. You can find it via genome repositories like NCBI and more specifically NCBI Genome Browser

Primer Design for EvaGreen Assays

For EvaGreen assays, simply order primers and the correct supermix. Optimize your ddPCR assays (e.g., thermal gradient testing) to ensure specificity and efficiency before using them on unknown samples.

Probe Design for Multiplexing

If using probes, Primer3Plus can generate probe sequences flanked by forward and reverse primers. Make sure to modify the following settings first:

Recommended Primer3Plus Settings:

Internal Oligo:

  • Set the oligo minimum to 15 bases.

  • Minimum probe Tm = 64°C, optimal Tm = 65°C, maximum Tm = 70°C (relax for high/low GC targets).

  • Oligo size: 13–30 nucleotides.

Also make sure to adhere to these Bio-Rad-issued guidelines:

  • Probe Tm should be 3–10°C higher than primers.

  • GC content: 30–80%. Design probes to favor Cs over Gs.

  • Length: <30 nucleotides (longer probes or Tm enhancers are recommended if to achieve the required Tm. I would recommend shorter probes that still satisfy the 1. and 2. points above).

  • Avoid G at the 5’ end (quenches fluorescence).

  • Label probes with compatible fluorophores (e.g., FAM, HEX/VIC = QX200 or FAM, HEX/VIC, Cy5, Cy5.5, ROX and ATTO 590 = QX600) and quenchers (e.g., IOWA BLACK).

Validating Primer and Probe Sequences

I. Self-Annealing and Structural Issues:

Use PCR Primer Stats to identify:

  • GC clamps
  • Self-annealing
  • Hairpin formation

A few self-annealing base pairs are manageable, but excessive structures can reduce reaction efficiency.

II. Primer-Dimer Analysis:

Check forward-reverse primer interactions using Multiple Primer Analyzer. Input your sequences in fasta format and adjust:

  • Primer concentration: 900 nM.
  • Probe concentration: 250 nM.

  • Salt concentration: 53.8 mM (50 mM monovalent, 3.8 mM divalent).

    This tool provides Tms, self-dimer stats, and other crucial metrics. Ensure primer Tms differ from probes by 3–10°C.

III. SNP Detection Probes Check-up:

For SNP assays, design two probes—one for wild type (e.g., AGG) and another for the mutant (e.g., ACG). Minimize cross-reactivity using Oligo Analyzer:

  • Input probe sequences individually.
  • Adjust oligo (0.25 μM), Na+ (50 mM), Mg++ (3.8 mM), and dNTP (0.8 mM) concentrations.
  • Click on TM MISMATCH (to the right)
  • Scroll down to the bottom, introduce a mismatch of your choosing in the probe sequence and click on CALCULATE at the bottom
  • Use Tm mismatches to evaluate the binding specificity of your probes and calculate the delta Tm between them (ideal: ≥2°C).

Place the SNP locus centrally in the probe sequence to maximize the mismatch’s effect.

Bio-Rad Custom Assay Builder

Simplify evaluations with Bio-Rad’s Custom Assay Builder. Enter your ddPCR platform, supermix, and sequences to receive a suitability report with optimization recommendations.

Final Steps

Once designed, ensure your primers and probes:

  • Follow recommended guidelines.
  • Have minimal structural issues (in silico).

Test them in vitro for final validation. This ensures optimal performance before applying them to unknown samples.