About KRAS
The KRAS gene (Kirsten rat sarcoma viral oncogene homolog) on chromosome 12p12.1 encodes a small GTPase that transduces signals from growth factor receptors. KRAS mutations, particularly in codons 12, 13, and 61, are among the most common oncogenic drivers in pancreatic, colorectal, and lung cancers. KRAS spans ~45 kb with 6 coding exons.
NCBI Gene ID: 3845 | RefSeq: NM_004985.5 | Genomic: NC_000012.12 (25205246-25250930)
KRAS Primer Design Challenges
- Hotspot codons 12 & 13: Exon 2 contains the most frequently mutated residues (G12C, G12D, G12V, G13D), requiring precise amplicon targeting
- GC-rich exon 2: Over 65% GC content around codons 12–13, requiring adjusted PCR parameters
- Pseudogenes: KRASP1 pseudogene shares high sequence homology with exons 4–6, risking non-specific amplification
- Small gene size: Only 6 exons with short introns limit primer placement options
- Low tumor content: Liquid biopsy applications require high sensitivity primers for detecting low-frequency mutations
Recommended Primer Design Parameters for KRAS
| Parameter | Exons 2, 3 (Hotspots) | Exons 4–6 |
|---|---|---|
| Primer length | 22-25 nt | 20-22 nt |
| GC content | 50-60% | 45-55% |
| Tm | 60-65°C | 58-62°C |
| Amplicon size | 120-250 bp | 150-300 bp |
| Annealing temp | 62-65°C | 58-60°C |
| PCR additive | 5% DMSO recommended | Standard |
Key SNPs to Avoid in Primer Binding Sites
When designing KRAS primers, avoid these clinically significant variants:
- rs121913530 (c.34G>T, G12C) — Most common KRAS mutation in NSCLC
- rs121913529 (c.35G>A, G12D) — Frequent in pancreatic cancer
- rs112445441 (c.38G>A, G13D) — Exon 2 codon 13 variant
- rs121913535 (c.37G>T, G13C) — Exon 2 codon 13 variant
- rs121913240 (c.182A>T, Q61L) — Exon 3 codon 61 mutation
Clinical Validation Required
All KRAS 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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