About PrimerGenie
PrimerGenie is a free PCR primer search tool built by Cytogence, the bioinformatics division of KeyQ.
Methodology
Primer Design
Primers are computationally designed from RefSeq-annotated transcripts downloaded from the UCSC Genome Browser. The design pipeline:
- Parses refGene annotations to extract exon structures for all coding transcripts
- Extracts spliced mRNA sequences (concatenated exons) from reference genomes
- Scans for primer pairs that span intron-exon junctions (inter-exonic design) to distinguish cDNA amplification from genomic DNA contamination
- Filters candidates by melting temperature (Tm), GC content, homopolymer runs, self-complementarity, and 3' primer-dimer potential
- Checks specificity by counting how many transcripts each primer pair could amplify using Aho-Corasick multi-pattern matching across the full transcriptome
Thermodynamics
Melting temperature is calculated using the SantaLucia nearest-neighbor method (SantaLucia 1998) with the following parameters:
- Nearest-neighbor enthalpy/entropy parameters from unified NN tables
- Salt correction for 50 mM Na⁺ (Owczarzy et al., 2004)
- Primer concentration of 250 nM
Default Filter Parameters
| Parameter | Default Value |
|---|---|
| Primer Length | 20 bp |
| Melting Temperature | 50–65 °C |
| GC Content | 50–65% |
| Max Homopolymer | 3 consecutive bases |
| Product Size | 80–300 bp |
| Max Tm Difference | 5 °C |
Specificity
A primer pair is considered specific (targets = 1) if it matches exactly one transcript in the reference transcriptome. Matching criteria: forward primer matches in the sense orientation, reverse primer (reverse complement) matches downstream within 500 bp.
Supported Species
Human
Assemblies: hg38 (default), hg19
Source: UCSC Genome Browser
Mouse
Assemblies: mm39 (default), mm10
Source: UCSC Genome Browser
Citing PrimerGenie
If you use PrimerGenie in your research, please cite:
PrimerGenie: A free PCR primer search tool with community validation. Cytogence, KeyQ. Available at: https://primergenie.com
References
- SantaLucia J Jr. (1998) "A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics." PNAS 95:1460-1465
- Owczarzy R, et al. (2004) "Effects of sodium ions on DNA duplex oligomers." Biochemistry 43:3537-3554
- UCSC Genome Browser: genome.ucsc.edu
Contact
For questions, feedback, or partnership inquiries, reach out to Cytogence.