About FLT3
The FLT3 gene (Fms Related Receptor Tyrosine Kinase 3) on chromosome 13q12 FLT3-ITD mutations in the juxtamembrane domain (exon 14–15) and TKD mutations at D835 (exon 20) are poor-prognosis markers in AML. Key targets include Exons 14–15 (JM domain), exon 20 (TKD) for comprehensive mutation screening.
Mutations: ITD (internal tandem duplication), D835Y, D835V | GC content: 48% | Target exons: Exons 14–15 (JM domain), exon 20 (TKD)
FLT3 Primer Design Challenges
- ITD size variability: FLT3-ITD insertions range from 3 to 400+ bp requiring fragment analysis or long-range PCR across the JM domain
- Dual mutation hotspots: JM domain (exons 14–15) and TKD (exon 20) require two completely different amplicon design strategies
- Lower GC content (48%): Moderate AT bias in intron 14 requires longer primers (24–26 nt) for specific binding
Recommended Primer Design Parameters for FLT3
| 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 FLT3
| Target Region | Forward Primer (5′→3′) | Reverse Primer (5′→3′) | Amplicon |
|---|---|---|---|
| Exons 14–15 (ITD) | 5′-TGTGCAATTCCAGACTCTGC-3′ | 5′-GCGACTTTGTGTGTGCTGAT-3′ | 230 bp |
| Exon 20 (D835) | 5′-CCAGCCACAGCATCAGTCAT-3′ | 5′-CATCCACCTCCCAACTGAAC-3′ | 198 bp |
Key SNPs to Avoid in Primer Binding Sites
When designing FLT3 primers, avoid these clinically significant variants:
- FLT3-ITD (ITD (variable)) — Exons 14–15 juxtamembrane duplication
- rs121913400 (D835Y) — Exon 20 TKD point mutation in AML
- rs121913401 (D835V) — Exon 20 TKD activating mutation
Clinical Validation Required
All FLT3 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 FLT3 Primers Now
Pre-configured with FLT3-specific parameters. Use our primer design, Tm calculator, and GC content tools for optimal results.