Design PCR primers for SNP genotyping
Back to ToolsEnter any target size you want. The PCR product cannot be longer than the DNA you paste above—there is no sequence beyond that. If your target is larger than the template, the tool still designs primers using the full pasted region (centered on the SNP). Minimum here is 30 bp (twice the minimum primer length).
This tool designs forward and reverse PCR primer sequences around a SNP position you specify, with simple Tm and GC% estimates. All logic runs in your browser. Use unambiguous A/T/G/C DNA only—no IUPAC codes.
Scope: Educational layout—simple Tm/GC rules, not Primer3 or commercial primer QC (no hairpin, dimer, or off-target checks). Validate primers in the lab before use. For research and teaching, not clinical or diagnostic workflows.
SNP genotyping is the process of determining which DNA sequence variants (alleles) are present at specific SNP positions in an individual's genome. PCR primers are needed to amplify the region containing the SNP before genotyping methods like Sanger sequencing, RFLP analysis, or allele-specific PCR can be applied. Well-designed primers ensure efficient and specific amplification of the target region.
Enter the SNP as a 0-based index in your sequence (after pasting, line breaks and FASTA > header lines are ignored). For example, the 21st base is index 20. The tool centers an amplicon of your chosen length on that index, then sets the forward primer to the first N bases at the 5' end of that amplicon and the reverse primer to the reverse complement of the last N bases at the 3' end, where N is your primer length. It does not search or tune primer sequences for Tm/GC—those values are computed for reporting only.
In the lab, many protocols use 18–25 nt primers; this tool lets you set primer length between 15 and 30 bases (default 20). Pick a length that fits your polymerase and assay; the app only uses that length to slice sequence—it does not rank alternative primers.
The optimal amplicon length depends on your specific application. For standard PCR and Sanger sequencing, amplicons of 150-500 bp generally work well. For allele-specific PCR or RFLP analysis, you might prefer shorter amplicons (100-300 bp). For next-generation sequencing applications, even shorter amplicons (70-200 bp) may be preferred. The default value of 200 bp is a good starting point for most applications. In this tool, the PCR product cannot be longer than the sequence you paste—if you request a larger target than your template length, the program uses the full pasted region as the effective amplicon (the form enforces a minimum of 30 bp).
GC% is the percentage of G and C bases in each primer sequence shown. Tm here is a quick estimate using the Wallace rule: Tm (°C) = 4 × (G + C) + 2 × (A + T) for that primer. It is a rough guide, not a nearest-neighbor or salt-corrected model. In practice, many users aim for similar Tm between the two primers (often within a few °C) and GC% in a moderate range—those are general PCR tips, not rules enforced by this tool.