About KIT
The KIT gene (KIT Proto-Oncogene) on chromosome 4q12 D816V in the kinase activation loop (exon 17) and exon 11 juxtamembrane mutations are therapeutic targets in GIST and systemic mastocytosis. Key targets include Exons 8–18 (JM domain, kinase domain, activation loop) for comprehensive mutation screening.
Mutations: D816V, D816H, V559D, W557-K558 del, N822K | GC content: 53% | Target exons: Exons 8–18 (JM domain, kinase domain, activation loop)
KIT Primer Design Challenges
- Multiple mutation hotspots: Exon 11 (JM domain), exon 13 (ATP pocket), exon 17 (activation loop) require three separate amplicons for full coverage
- Moderate GC (53%): Balanced GC with localized high-GC regions in exons 11 and 17 requiring Tm optimization
- Drug resistance mutations: Secondary T670I and D816V mutations emerge during imatinib/sunitinib therapy requiring sensitive detection
Recommended Primer Design Parameters for KIT
| Parameter | Standard Exons | Optimized Regions |
|---|---|---|
| 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 (touchdown) |
| PCR additive | Standard | Optional 3–5% DMSO for problematic regions |
Recommended Primer Sequences for KIT
| Target Region | Forward Primer (5′→3′) | Reverse Primer (5′→3′) | Amplicon |
|---|---|---|---|
| Exon 11 (JM domain) | 5′-TGACCTGCATGCGACATCAT-3′ | 5′-ACTGTGGTCCTTGAAGCACA-3′ | 215 bp |
| Exon 17 (D816 region) | 5′-GCCGCTGTGTTACGATCTTC-3′ | 5′-GGCAAGAGAGCAATGACTCC-3′ | 198 bp |
Key SNPs to Avoid in Primer Binding Sites
When designing KIT primers, avoid these clinically significant variants:
- rs121913507 (D816V) — Exon 17 activation loop in mastocytosis
- rs121913517 (V559D) — Exon 11 juxtamembrane GIST mutation
- rs3822214 (M541L) — Exon 10 common polymorphism
Clinical Validation Required
All KIT 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.
Design KIT Primers Now
Pre-configured with KIT-specific parameters. Use our primer design, Tm calculator, and GC content tools for optimal results.