About BRAF
The BRAF gene (B-Raf proto-oncogene) on chromosome 7q34 encodes a serine/threonine-protein kinase in the MAPK/ERK signaling pathway. The BRAF V600E mutation (c.1799T>A) is the most common activating mutation, found in ~50% of melanomas, ~10% of colorectal cancers, and ~45% of papillary thyroid carcinomas. BRAF spans ~190 kb with 18 coding exons.
NCBI Gene ID: 673 | RefSeq: NM_004333.6 | Genomic: NC_000007.14 (140719327-140924764)
BRAF Primer Design Challenges
- V600E hotspot in exon 15: The most common mutation site requires precise amplicon placement around codon 600
- GC-rich exon 15: Approximately 65% GC content around the V600E mutation site
- Large introns: Introns 8 and 10 span>20 kb, requiring careful intron-spanning primer design for cDNA applications
- Alternative splicing: Multiple BRAF transcripts (including truncated isoforms) require transcript-aware design
- Pseudogene interference: BRAF pseudogene (BRAFP1) shares partial sequence homology with exons 4–11
Recommended Primer Design Parameters for BRAF
| Parameter | Standard Exons | GC-Rich Exons (11, 15) |
|---|---|---|
| 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 | 62-65°C |
| PCR additive | Standard | Add 5% DMSO or 1M betaine |
Key SNPs to Avoid in Primer Binding Sites
When designing BRAF primers, avoid these clinically significant variants:
- rs113488022 (c.1799T>A, V600E) — Most common BRAF activating mutation in melanoma
- rs397516897 (c.1799_1801delTGA, V600_K601delinsE) — Exon 15 in-frame deletion-ins
- rs121913351 (c.1397G>T, G466V) — Exon 11 kinase domain variant
- rs121913370 (c.1742A>G, N581S) — Exon 15 mutation
- rs121913355 (c.1406G>C, G469A) — Exon 11 non-V600 mutation
Clinical Validation Required
All BRAF 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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