Design PCR primers for DNA sequences with Primer3 controls for regions, Tm, GC content, and product size. Learn more
Primer3 webserver overview
Primer3 is one of the standard tools for PCR primer design. It searches a DNA template for primer pairs that satisfy thermodynamic, compositional, positional, and product-size constraints, then ranks the surviving candidates by penalty score.
ProteinIQ runs the native Primer3 2.6.1 core. The tool keeps Primer3's default generic PCR workflow and supports a focused set of native controls without reimplementing the scoring model.
For related preprocessing, you can use GC Content to inspect template composition or DNA to Protein to check coding sequence context before primer design.
For a step-by-step workflow covering region controls, Tm and GC limits, complementarity checks, and specificity validation, see How to use Primer3 online.
How Primer3 chooses primers
Primer3 evaluates candidate oligos against hard constraints such as:
- primer length
- melting temperature ()
- GC content
- self-complementarity and 3' complementarity
- product size
- allowed or excluded template regions
Candidates that pass those filters are ranked by penalty score. Lower penalties are better and indicate candidates that sit closer to the configured optima.
Inputs
| Input | Accepted format | Limits and behavior |
|---|---|---|
DNA Sequence | Raw DNA sequence or single-record FASTA text, .fasta, .fa, .fas, .txt, or .seq file | One required DNA sequence, up to 10 MB. Multi-record FASTA is rejected instead of silently using only the first record. |
Job name | Text | Optional label stored with the run. |
Settings
Main settings stays visible for the standard design workflow. Advanced settings is collapsed by default and contains optional controls for primer design, primer quality, secondary structure, PCR chemistry, 5′ overhangs, and result diagnostics. Dependent fields appear only when their parent task or configuration section is enabled.
Main settings
| Setting | Default or range | Description |
|---|---|---|
Task | Design primer pairs | Runs Primer3's generic task for new primer pairs or Check existing primers for the native check_primers task. |
Left primer (5' -> 3') | Not set | Optional forward primer used only with Check existing primers. Provide it in the 5′ to 3′ direction. |
Right primer (5' -> 3') | Not set | Optional reverse primer used only with Check existing primers. Provide it in the 5′ to 3′ direction; ProteinIQ maps it to Primer3's reverse-complement input. |
Check behavior | Off | Opens controls for how provided primers that fail hard constraints are handled. Available with Check existing primers. |
Return primers that fail constraints | Off | Enables PRIMER_PICK_ANYWAY so failing provided primers are returned with native problem descriptions. |
Region constraints | Off | Opens target, included, and excluded region controls. |
Region mode | Auto | Selects Auto with no region constraint, Target region mapped to SEQUENCE_TARGET, or Included region mapped to SEQUENCE_INCLUDED_REGION. |
Region start position | 1; 1-1,000,000 | 1-based start for the target or included region. Available for Target region and Included region. |
Region length (bp) | 100; 1-100,000 | Length of the target or included region. Available for Target region and Included region. |
Excluded region start | Not set; 1-1,000,000 | Optional 1-based start of a region that primers cannot overlap. Use it with Excluded region length. |
Excluded region length (bp) | Not set; 1-100,000 | Optional length of the excluded region, mapped to SEQUENCE_EXCLUDED_REGION. |
Minimum Tm (C) | 57°C; 40-80°C | Minimum acceptable melting temperature. |
Maximum Tm (C) | 63°C; 55-85°C | Maximum acceptable melting temperature. |
Minimum product size (bp) | 100; 1-2,000 | Minimum acceptable PCR product size. |
Maximum product size (bp) | 300; 1-5,000 | Maximum acceptable PCR product size. |
Number of primer pairs | 5; 1-20 | Number of primer pairs returned by the run. |
Primer design
| Setting | Default or range | Description |
|---|---|---|
Primer design | Off | Opens primer length, optimal Tm, GC, and product-size preferences. |
Optimal Tm (C) | 60°C; 50-75°C | Target melting temperature used when ranking primers against the selected optimum. |
Minimum primer size (bp) | 18; 10-35 | Minimum primer length. |
Optimal primer size (bp) | 20; 15-30 | Target primer length used for ranking. |
Maximum primer size (bp) | 27; 18-36 | Maximum primer length. Primer3 accepts values up to 36 in this interface. |
Minimum GC (%) | 20; 0-100 | Minimum primer GC percentage. |
Optimal GC (%) | Not set; native default 50 | Optional target GC percentage used for ranking. |
Maximum GC (%) | 80; 0-100 | Maximum primer GC percentage. |
Optimal product size (bp) | Not set; 1-5,000 | Optional target product size used for ranking. It does not replace the minimum and maximum product-size constraints. |
Additional product size ranges (bp) | Not set | Optional comma- or semicolon-separated intervals such as 400-500, 600-700. A product in any listed interval is accepted. |
Primer quality
| Setting | Default or range | Description |
|---|---|---|
Primer quality | Off | Opens homopolymer, GC-clamp, ambiguous-base, and primer-pair Tm controls. |
Max homopolymer length | 5; 1-10 | Maximum length of a mononucleotide repeat in a primer, mapped to PRIMER_MAX_POLY_X. |
GC clamp (3' end) | 0; 0-5 | Requires this many G or C nucleotides at the 3′ end of each primer. |
Max GC at 3' end | 5; 0-5 | Maximum number of G or C nucleotides in the last five bases at a primer's 3′ end. |
Max Ns accepted | 0; 0-5 | Maximum number of unknown bases, N, allowed in a primer. |
Max Tm difference (C) | 100°C; 0-100°C | Maximum allowed Tm difference between the left and right primers. |
Secondary structure
| Setting | Default or range | Description |
|---|---|---|
Secondary structure | Off | Opens self-complementarity, pair-complementarity, hairpin, and 3′ stability controls. |
Max 3' end stability (kcal/mol) | Not set; native default 100 | Optional cap on the stability of the last five 3′ bases. The native default is effectively off. |
Max self-complementarity Tm (C) | 47°C; 0-100°C | Maximum thermodynamic Tm for a primer pairing with another copy of itself. |
Max 3' self-complementarity Tm (C) | 47°C; 0-100°C | Maximum thermodynamic Tm for a self-complementary interaction anchored at a primer's 3′ end. |
Max pair complementarity Tm (C) | 47°C; 0-100°C | Maximum thermodynamic Tm for complementarity between the left and right primers. |
Max 3' pair complementarity Tm (C) | 47°C; 0-100°C | Maximum thermodynamic Tm for left and right primer complementarity anchored at a 3′ end. |
Max hairpin Tm (C) | Not set; native default 47°C | Optional maximum melting temperature for an intramolecular primer hairpin. |
Primer3 evaluates these complementarity limits with its thermodynamic oligo-alignment mode. The limits are maximum duplex or hairpin melting temperatures, not percentages.
PCR chemistry
| Setting | Default or range | Description |
|---|---|---|
PCR chemistry | Off | Opens thermodynamic method, salt, primer concentration, annealing temperature, and additive controls. |
Tm calculation method | SantaLucia 1998 | Selects SantaLucia 1998 or Breslauer 1986 for the thermodynamic Tm table. |
Salt correction formula | SantaLucia 1998 | Selects SantaLucia 1998, Schildkraut-Lifson 1965, or Owczarzy 2004 for salt correction. |
Salt concentration (mM) | 50; 0.001-500 | Monovalent salt concentration used for Tm calculation. |
Divalent cation concentration (mM) | 1.5; 0-100 | Divalent cation concentration, such as Mg²⁺, used for Tm calculation. |
dNTP concentration (mM) | 0.6; 0-10 | Total dNTP concentration used for Tm calculation. |
DNA concentration (nM) | 50; 0.001-1,000 | Primer concentration used for Tm calculation. |
Annealing temperature (C) | Not set; 0-100°C | Optional annealing temperature used to calculate bound-percentage metrics. It cannot be combined with the Owczarzy 2004 salt correction. |
DMSO concentration (%) | Not set; native default 0; 0-100 | Optional DMSO concentration used in the Tm correction. |
DMSO factor | Not set; native default 0.6; 0-10 | Optional DMSO correction factor. |
Formamide concentration (mol/L) | Not set; native default 0; 0-20 | Optional formamide concentration used in Tm calculation. |
5′ overhangs
| Setting | Default or range | Description |
|---|---|---|
5′ overhangs | Off | Opens optional left and right 5′ overhang sequences. |
Left primer 5' overhang | Not set | Optional sequence added to the 5′ end of the left primer through SEQUENCE_OVERHANG_LEFT. |
Right primer 5' overhang | Not set | Optional sequence added to the 5′ end of the right primer through SEQUENCE_OVERHANG_RIGHT. |
Primer3 includes overhangs in complementarity and product-size calculations where its native behavior defines them. Tm and GC percentage remain tied to the template-binding portion.
Result diagnostics
| Setting | Default or range | Description |
|---|---|---|
Result diagnostics | Off | Opens the optional Primer3 explanation fields. |
Include Primer3 diagnostics | Off | Returns considered, rejected, and accepted primer counts and other native explanation fields. |
Understanding the results
The results table is built from native Primer3 output. ProteinIQ keeps the original tag map in the result payload and also presents a spreadsheet view with high-value fields such as:
- primer sequences
- derived 1-based binding ranges
- native Primer3 coordinate tuples
- Tm and GC%
- product size
- pair penalty
- left and right penalties
- pair and 3' pair complementarity Tm
- self-complementarity and 3' self-complementarity Tm per primer
- hairpin Tm per primer
- end stability
- bound percentage when available
- native left and right problem descriptions when failing primers are returned
Primer3's explain strings are returned alongside the table so you can see why candidates were rejected or why not enough primer pairs were found.
Notes and limits
- this tool uses Primer3's generic primer-design task and its
check_primerstask. It does not currently support the broader task families such as sequencing primers or internal oligo (hybridization probe) workflows. - Primer3 optimizes against the submitted template only. It does not check whole-genome off-target binding.
- Restriction sites, SNP-aware specificity, and external genome-level validation should be checked with downstream tools such as Primer-BLAST when needed.
