Analyze primer self-dimers and cross-dimers in seconds accurately. Tune thresholds, salt, and 3′ penalties easily. Design cleaner primers, speed optimization, and improve amplification success.
| Forward (5′→3′) | Reverse (5′→3′) | Typical goal |
|---|---|---|
| AGTCCGATGCTAGTACG | CCGTATCGTACGATGGA | Low 3′ complement and balanced Tm |
| GCGTACGATCGATCGTT | AACGATCGATCGTACGC | Avoid long complementary stretches |
This calculator scans every possible alignment between a primer and the reverse complement of its partner. It reports the longest contiguous complementary run and a stricter run that ends at the forward primer 3′ base, where polymerase extension begins. In routine PCR, a 3′ run of four or more bases is often a practical warning sign. It is designed for quick screening before ordering primers or redesigning projects.
Internal complementarity can form transient duplexes without creating an extendable end. In contrast, complementarity at the last bases can generate an extendable primer–primer template and consume reagents. The adjustable 3′ window highlights stability near the forward primer end, helping you prioritize edits that remove risky terminal matches.
For many assays, primers of 18–25 bases with 40–60% GC provide robust binding while limiting secondary structure. A quick melting estimate is provided using the Wallace rule, which is most reliable for shorter oligos. Large Tm gaps between primers can increase non‑specific products even if dimer risk is low. Aim for a Tm difference within 2–3 °C when possible and avoid extreme GC clamps.
To give an easy stability proxy, matched GC pairs contribute −1.5 and matched AT pairs contribute −1.0 to a heuristic ΔG total across the best alignment. More negative values suggest stronger duplex formation. This score is not a nearest‑neighbor thermodynamic calculation, but it helps rank candidate primer pairs consistently. Use it to compare alternatives after you break a risky 3′ match.
Use the Min contiguous run and Min 3′ run fields to reflect your lab’s tolerance. For high‑sensitivity qPCR, many teams set the 3′ threshold at 4 and the overall run threshold at 6–8. For endpoint PCR with higher primer concentrations, you may choose stricter limits and redesign aggressively. If you see recurring dimers, raise thresholds and shorten primers by one or two bases.
After calculation, the results panel summarizes forward self‑dimer, reverse self‑dimer, and cross‑dimer risk and visualizes the key values in an interactive Plotly graph. Export to CSV for batch documentation or to PDF for a clean attachment in validation notes. Iterate by changing one end base at a time and rechecking the 3′ run.
A primer dimer is a primer–primer duplex formed by complementarity, sometimes extendable by polymerase. It can consume primers and nucleotides, reducing target amplification and creating unwanted bands or qPCR signal.
Prioritize the Max 3′ run, because terminal complementarity can be extended. A modest internal match may be tolerable, but a strong 3′ match often creates dominant dimers, especially in early cycles.
No. The ΔG value here is a screening proxy that weights GC pairs more than AT pairs. For publication‑grade thermodynamics, use nearest‑neighbor models with salt and oligo concentration parameters.
Many workflows target 18–25 bases, 40–60% GC, and a similar Tm for both primers. Avoid long complementary stretches and extreme GC clamps at the 3′ end when possible.
Change one or two bases near the 3′ end to break contiguous complementarity, or shorten the primer by 1–3 bases. Recheck Max 3′ run after each edit and keep primer Tm balanced.
Dimers can be promoted by high primer concentration, low annealing temperature, or complex templates. Consider raising annealing temperature, lowering primer concentration, and verifying magnesium and cycling conditions alongside sequence improvements.
Important Note: All the Calculators listed in this site are for educational purpose only and we do not guarentee the accuracy of results. Please do consult with other sources as well.