Pimers (Re)Design
In some cases the switch from gel electrophoresis to HRM requires primers redesign.
To be on the safe side it is recommended to design separate reaction for WT and separate reaction for the target gene (mutant).
Here are the guidelines of Applied Biosystems for optimal Primers design:
- Use amplicons with a target length of <300 (250) bp to ensure sensitive detection of sequence variants
- Optimally, design three primer sets to empirically determine the most robust combination
Avoid consecutive identical nucleotides. If you are unable to avoid consecutive identical nucleotides, make sure that each primer contains fewer than 4 consecutive Gs.
| Design attribute | Design guidelines |
|---|---|
| Amplicon | Length is less than 250 basepairs |
| Primer length | ~20 bases each |
| Tm | 58 °C to 60 °C (Optimal Tm is 59 °C) |
| % GC content | 30–80% GC content in each primer |
| 3’ end | No more than 2 G+C residues in the last 5 nucleotides at the3′ end |
| Repeating oligonucleotides |