PrimerDesigner Expert — Diseño de primers para secuenciación Sanger

Parameters

Target sequence (FASTA or plain)

Search regions

Forward (from – to)

Reverse (from – to)

or use own Forward

or use own Reverse

General criteria

Primer length (min – max)

Tm range (°C)

Opt Tm (°C)

Max ΔTm pair (°C)

GC% (min – max)

GC clamp (min)

Min quality score

Amplified fragment size

Min – Max (nt)

Best pairs to return

Advanced

Max 3' stability score

Max poly-X run

Max self-end dimer (Tm)

[Na⁺] mM

[Mg²⁺] mM

[oligo] nM

[dNTP] mM

Min SNP dist (nt) Sanger

Results: — pairs found

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Enter sequence and click Find Primers

#	Forward primer 5'→3'	Pos	Tm	Q	Reverse primer 5'→3'	Pos	Tm	Q	Amp (nt)	Q pair	Dist FWD→SNP	Dist SNP→REV

Select a primer pair to view details

No selection

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PCR in silico — test primer pair on target sequence

Forward primer (5'→3')

Reverse primer (5'→3' minus strand)

Variant position in sequence (optional)

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Enter primer pair and run PCR in silico Uses sequence from Design tab · Specify variant position to check Sanger readability

— Click a row to edit notes inline

Type	Gene / Pair ID	Sequence 5'→3'	Tm (°C)	GC%	Len	Q	Amp (nt)	Notes	Date

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No primers saved yet Select pairs in Design tab and click Save to DB

How it works / Cómo funciona

Tm. Nearest-neighbor method of SantaLucia 1998 with monovalent/Mg²⁺ salt correction of Owczarzy 2004 (Mg²⁺ chelated by dNTPs is discounted). Valid for oligonucleotides; matches Primer3/Primer-BLAST within the usual range.

Search regions. This is what distinguishes the expert version: forward and reverse are constrained to independent windows (from – to, 1-based). Use it when the template has fixed anchors — a known exon, a conserved region, or a sequence you must not prime on. Leave empty to search the whole template.

Sanger readability. If a variant position is given, the pair must place it inside the amplicon and at least the minimum distance from each primer (default 60 nt), because the first ~40–60 nt after the primer fall in the noisy region of the electropherogram. Distances FWD→SNP and SNP→REV are reported per pair.

Annealing temperature (Ta). The optimal Ta of Rychlik 1990 is reported: 0.3·Tm(least stable primer) + 0.7·Tm(product) − 14.9, using the nearest-neighbor Tm of the primer and the product Tm (81.5 + 16.6·log₁₀[Na⁺] + 0.41·%GC − 675/N). It is more reliable than the plain "Tm − 5" rule, especially with long amplicons or genomic DNA; the authors reported agreement with experimental Ta within 0.7 °C. The −5 rule is shown as a reference only.

IUPAC. Degenerate bases are accepted in the template (flagged in amber), but primers are never designed over degenerate positions.

Privacy. Everything runs in your browser. The oligo database lives in this browser's IndexedDB and is never uploaded. This page loads no external fonts or libraries. The only outbound requests are the ones you trigger yourself by clicking a Primer-BLAST link.

Not included. This tool does not do qPCR design, probe design or cloning. Those options are absent, not hidden — do not assume defaults cover them.

Support tool. Always verify specificity in Primer-BLAST (links are provided per primer and per pair).

References / Referencias

SantaLucia J. Proc Natl Acad Sci USA 1998;95(4):1460-5. DOI: 10.1073/pnas.95.4.1460
Owczarzy R, You Y, Moreira BG, et al. Biochemistry 2004;43(12):3537-54. DOI: 10.1021/bi034621r
Rychlik W, Spencer WJ, Rhoads RE. Nucleic Acids Res 1990;18(21):6409-12. DOI: 10.1093/nar/18.21.6409