About CALR
The CALR gene (Calreticulin) on chromosome 19p13 Somatic frameshift mutations in exon 9 generate a novel C-terminal peptide that constitutively activates the thrombopoietin receptor MPL in myeloproliferative neoplasms. Key targets include Exon 9 for comprehensive mutation screening.
Mutations: Type 1 (52 bp del), Type 2 (5 bp ins) exon 9 mutations | GC content: 64% | Target exons: Exon 9
CALR Primer Design Challenges
- Very high GC content (64%): CALR has elevated GC content, particularly in exon 9 where all mutations cluster
- Mutation hotspot in exon 9: Type 1 (52 bp deletion) and Type 2 (5 bp insertion) share a common breakpoint region requiring carefully designed flanking primers
- Single-exon mutation pattern: All pathogenic mutations are limited to the last exon, allowing focused but high-resolution amplicon design
Recommended Primer Design Parameters for CALR
| Parameter | Standard Exons | GC-Rich Regions (≥58%) |
|---|---|---|
| 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 | Add 5–10% DMSO or betaine |
Recommended Primer Sequences for CALR
| Target Region | Forward Primer (5′→3′) | Reverse Primer (5′→3′) | Amplicon |
|---|---|---|---|
| Exon 9 (Type 1/2) | 5′-GAAGCAGCAGAAACGCACAA-3′ | 5′-TCAGCTCCTGGACAGAGTCA-3′ | 198 bp |
| Exon 9 alternate | 5′-GGCCTTGTCCAGTTCCACAA-3′ | 5′-TGCCTCCTGTCTTGTGATGT-3′ | 165 bp |
Key SNPs to Avoid in Primer Binding Sites
When designing CALR primers, avoid these clinically significant variants:
- rs1554100933 (Type 1 (52 bp del)) — Exon 9 frameshift in essential thrombocythemia
- rs1554100938 (Type 2 (5 bp ins)) — Exon 9 insertion in primary myelofibrosis
Clinical Validation Required
All CALR 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 CALR Primers Now
Pre-configured with CALR-specific parameters. Use our primer design, Tm calculator, and GC content tools for optimal results.