About PrimerGenie

Methodology

Primer Design

Primers are computationally designed from RefSeq-annotated transcripts downloaded from the UCSC Genome Browser. The design pipeline:

1. Parses refGene annotations to extract exon structures for all coding transcripts
2. Extracts spliced mRNA sequences (concatenated exons) from reference genomes
3. Scans for primer pair candidates within the target product size range (70–300 bp)
4. Filters candidates by melting temperature (Tm), GC content, homopolymer runs, self-complementarity, and 3' primer-dimer potential
5. Scores and ranks primers by quality (see Primer Scoring below)
6. Checks specificity by counting how many transcripts each primer pair could amplify using Aho-Corasick multi-pattern matching across the full transcriptome

Inter-Exonic Design

For multi-exon genes, primers that span intron-exon boundaries are strongly preferred. These inter-exonic primers will only amplify cDNA (processed mRNA), not genomic DNA, making them ideal for qRT-PCR experiments where genomic DNA contamination is a concern.

Intron-spanning primers receive a significant scoring bonus. However, the pipeline does not require intron spanning as a hard filter — primers within the same exon are still included but ranked lower.

Single-Exon Genes

Some genes consist of only a single exon (e.g., certain histone genes, olfactory receptors). PrimerGenie designs primers for these genes as well. Single-exon primers are flagged in the output and cannot span introns by definition. Researchers using these primers should be aware that they will also amplify genomic DNA and may need to include DNase treatment or no-RT controls in their experimental design.

Thermodynamics

Melting temperature is calculated using the SantaLucia nearest-neighbor method (SantaLucia 1998) with the Owczarzy et al. (2008) salt correction for divalent cations. Conditions match standard PCR:

• Nearest-neighbor enthalpy/entropy parameters from unified NN tables
• Monovalent cation correction: 50 mM Na⁺/K⁺ (Owczarzy et al., 2004)
• Divalent cation correction: 1.5 mM Mg²⁺ (Owczarzy et al., 2008)
• dNTP concentration: 0.8 mM (chelates free Mg²⁺)
• Primer concentration: 250 nM

These conditions match the defaults used by NCBI Primer-BLAST. Tm values are typically within ±1-2°C of NCBI. Each primer card includes a link to validate in NCBI Primer-BLAST for direct comparison.

Default Filter Parameters

Primer Scoring

Each primer pair receives a quality score based on multiple criteria. Primers are ranked by score, with the top 10 per transcript retained. The scoring system prioritizes qRT-PCR suitability:

Specificity

Specificity is reported at two levels:

• Gene-level: A primer pair is gene-specific if it matches transcripts from only one gene. For example, a TP53 primer that hits 15 TP53 transcript variants but no other genes is gene-specific.
• Transcript-level: The total number of transcripts matched across the transcriptome, including multiple isoforms of the same gene.

Specificity checking uses Aho-Corasick multi-pattern matching with overlapping detection to scan all primer sequences against every coding transcript simultaneously. Matching criteria: forward primer matches in the sense orientation, reverse primer (reverse complement) matches downstream, and the distance between them is within 500 bp on the spliced mRNA.

Output Fields

Supported Species

Citing PrimerGenie

If you use PrimerGenie in your research, please cite:

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
• Owczarzy R, et al. (2008) "Predicting stability of DNA duplexes in solutions containing magnesium and monovalent cations." Biochemistry 47:5336-5353
• UCSC Genome Browser: genome.ucsc.edu

Contact

For questions, feedback, or partnership inquiries, reach out to Cytogence.