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PCR, qPCR & Molecular Biology

Primer-Pair Sequence-Property Comparison Workbench

Compare two exact DNA oligonucleotides under one declared nearest-neighbour sodium model and inspect exact reverse-complement tracts without issuing a compatibility verdict or annealing-temperature prescription.

Biology · experimental measurements

Keep strand melting estimates, GC composition and literal between-primer complementarity visible while leaving specificity, secondary-structure energy and assay validation to appropriate sequence and experimental tools.

Private calculations in your browser · explicit inputs and model boundaries
Example preview · Balanced pairConditional strand melting estimates
Forward primer56.1797859 °C
Reverse primer55.867004 °C
-56.1797859056.1797859

Both bars use the same perfect-match DNA/DNA concentration and sodium convention. Their separation is a model comparison, not an assay-compatibility grade.

  1. 1EnterProvide the known values
  2. 2CalculateResults update automatically
  3. 3VerifyReview the details and units
Try an example

Enter values in nM.

This named correction does not infer magnesium, dNTP or mixed-ion effects.

Calculation result

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Understand the relationship

The reasoning behind the result

Two melting estimates share one declared model

ΔTm = |Tm,F − Tm,R|

A difference is meaningful only under the same concentration, sodium and perfect-match convention.

The workbench reports the difference without applying a universal acceptable range.

Literal complementarity is a sequence observation

tract = longest exact A ↔ reverse-complement(B) run

An exact run can be located without assigning a free energy or predicting whether a dimer forms.

The separate both-3′ row identifies only a tract that reaches both written 3′ ends.

Compatibility requires more evidence

Hairpins, internal loops, mismatches, magnesium, concentration and reaction chemistry require a fuller structure model.

Target specificity requires a sequence-database or whole-template search and experimental confirmation.

Follow the numbers

Compare a declared primer pair

  1. Normalize both exact DNA records.
  2. Calculate each conditional perfect-match strand Tm under the same entered solution model.
  3. Align one record against the reverse complement of the other at every offset.
  4. Retain the longest exact tract and the longest tract reaching both 3′ ends.

The output is an inspectable property comparison, not an assay design approval.

Quick guide

How to use this calculator

  1. Enter both primers in their written 5′→3′ direction.
  2. Use the same explicit total strand and sodium concentrations for the two conditional Tm estimates.
  3. Inspect exact tracts and their coordinates; use an appropriate thermodynamic structure model and target-database search before experimental decisions.

Calculation method

Calculation and interpretation

Keep strand melting estimates, GC composition and literal between-primer complementarity visible while leaving specificity, secondary-structure energy and assay validation to appropriate sequence and experimental tools.

Each strand uses the named SantaLucia perfect-match DNA/DNA Tm model; exact complement tracts are longest common substrings between one primer and the reverse complement of the other.

Worked example

Compare a declared primer pair

The output is an inspectable property comparison, not an assay design approval.

Each strand uses the named SantaLucia perfect-match DNA/DNA Tm model; exact complement tracts are longest common substrings between one primer and the reverse complement of the other.

Supported inputs

Precision and limits

Exact DNA only

Each primer supports 2–60 canonical A/C/G/T bases; ambiguity, modifications and pre-existing gaps are excluded.

Sodium model only

The two Tm estimates use the same entered sodium-only correction and do not infer magnesium or mixed-ion conditions.

No dimer free energy

Exact tract length is not a hairpin or primer-dimer ΔG calculation and receives no universal risk threshold.

No specificity search

The workbench does not search a genome, transcriptome, plasmid, contaminant database or off-target space.

No annealing prescription

The result does not select a PCR annealing temperature or establish yield, efficiency or specificity.

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