Primer3

Design PCR primers for DNA sequences with scientifically faithful Primer3 controls for regions, Tm, GC content, and product size.

Input

DNA Sequence

Number of primer pairs

5 credits

Output

Configure inputs to begin

Set options on the left, then click “Design Primers”.

What is Primer3?

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 wraps 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.

How Primer3 chooses primers

Primer3 evaluates candidate oligos against hard constraints such as:

primer length
melting temperature ( $T_m$ )
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.

Input and settings

Sequence input

Submit one DNA sequence per job as raw text or a single-record FASTA file. Multi-record FASTA is rejected instead of silently using only the first record.

Region constraints

The Region mode control maps directly to Primer3's native sequence tags:

Auto: no region constraint
Target region: mapped to SEQUENCE_TARGET
Included region: mapped to SEQUENCE_INCLUDED_REGION

Use Region start position and Region length with either target or included mode. Excluded region start and Excluded region length map to SEQUENCE_EXCLUDED_REGION.

Core primer controls

Primer Size: maps to PRIMER_MIN_SIZE, PRIMER_OPT_SIZE, and PRIMER_MAX_SIZE
Melting Temperature: maps to PRIMER_MIN_TM, PRIMER_OPT_TM, and PRIMER_MAX_TM
GC Content: maps to PRIMER_MIN_GC, PRIMER_MAX_GC, and optionally PRIMER_OPT_GC_PERCENT
Product Size: maps to PRIMER_PRODUCT_SIZE_RANGE and optionally PRIMER_PRODUCT_OPT_SIZE

Quality and complementarity

Max homopolymer length: PRIMER_MAX_POLY_X
GC clamp: PRIMER_GC_CLAMP
Max GC at 3' end: PRIMER_MAX_END_GC
Max Ns accepted: PRIMER_MAX_NS_ACCEPTED
Max Tm difference: PRIMER_PAIR_MAX_DIFF_TM
Max self-complementarity: PRIMER_MAX_SELF_ANY
Max 3' self-complementarity: PRIMER_MAX_SELF_END
Max pair complementarity: PRIMER_PAIR_MAX_COMPL_ANY
Max 3' pair complementarity: PRIMER_PAIR_MAX_COMPL_END
Max hairpin Tm: PRIMER_MAX_HAIRPIN_TH

PCR chemistry and thermodynamics

The thermodynamics section maps to Primer3's chemistry-aware settings, including:

PRIMER_ANNEALING_TEMP
PRIMER_DMSO_CONC
PRIMER_DMSO_FACTOR
PRIMER_FORMAMIDE_CONC
PRIMER_SALT_MONOVALENT
PRIMER_SALT_DIVALENT
PRIMER_DNTP_CONC
PRIMER_DNA_CONC
PRIMER_TM_FORMULA
PRIMER_SALT_CORRECTIONS

If you set annealing temperature, Primer3 can also report bound-percentage metrics for returned primers.

5' overhangs

Optional left and right 5' overhangs map to SEQUENCE_OVERHANG_LEFT and SEQUENCE_OVERHANG_RIGHT. Primer3 includes these overhangs in complementarity and product-size calculations where native defines them, while Tm and GC% remain tied to the template-binding portion.

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 complementarity
3' pair complementarity
end stability
bound percentage when available

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 PCR-primer task. It does not currently support the broader task families such as sequencing primers or internal oligo 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.

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