About EGFR
The EGFR gene (Epidermal Growth Factor Receptor, also known as ErbB1/HER1) on chromosome 7p12 encodes a transmembrane tyrosine kinase receptor involved in cell proliferation and survival. EGFR mutations, particularly in exons 18–21, are key drivers in non-small cell lung cancer (NSCLC). EGFR spans ~190 kb with 28 coding exons.
NCBI Gene ID: 1956 | RefSeq: NM_005228.5 | Genomic: NC_000007.14 (55086724-55275031)
EGFR Primer Design Challenges
- Hotspot mutation regions: Exons 18–21 contain common activating mutations (L858R, exon 19 deletions, T790M) requiring targeted amplicons
- GC-rich promoter: The 5' regulatory region has>70% GC content
- Large introns: Intron 1 alone spans ~120 kb, requiring careful exon-specific primer placement
- Alternative splicing: Multiple EGFR isoforms necessitate transcript-aware primer design
- Pseudogenes: EGFR pseudogene on chromosome 7 can interfere with genomic amplification
Recommended Primer Design Parameters for EGFR
| Parameter | Standard Exons | GC-Rich / Hotspot Exons (18–21) |
|---|---|---|
| Primer length | 20-22 nt | 22-25 nt |
| GC content | 45-55% | 50-60% |
| Tm | 58-62°C | 60-65°C |
| Amplicon size | 150-300 bp | 180-350 bp |
| Annealing temp | 58-60°C | 60-64°C |
| PCR additive | Standard | Add 5% DMSO for GC-rich exons |
Key SNPs to Avoid in Primer Binding Sites
When designing EGFR primers, avoid these clinically significant variants:
- rs121434568 (c.2573T>G, L858R) — Common lung cancer mutation in exon 21
- rs121434569 (c.2369C>T, T790M) — Resistance mutation in exon 20
- rs28929495 (c.2155G>T, G719C) — Exon 18 activating mutation
- Exon 19 deletions (c.2235_2249del15) — Common in-frame deletions in NSCLC
Clinical Validation Required
All EGFR primers designed with VigyanLLM are for research use only. Clinical diagnostic applications require additional wet-lab validation, Sanger sequencing confirmation, and regulatory approval before patient use.
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