Primer3 operates by evaluating a target DNA sequence against a comprehensive set of user-defined constraints. The software suggests primer pairs based on several critical thermodynamic and physical parameters: Primer3—new capabilities and interfaces - PMC - NIH
To design multiplex primers using Primer 3, users simply need to:
| Parameter | Description | Example | |-----------|-------------|---------| | | Optimal length (18–27 nt) | Opt: 20 nt | | Product size range | Amplicon length (70–1000 bp) | Min 100, Max 300 bp | | Melting temperature (Tm) | Usually 50–65°C, max ΔTm ≤ 3°C | Opt: 60°C | | GC content | 40–60% recommended | Opt: 50% | | 3’-end stability | Avoid G or C in last 3–5 bases | Max poly-X = 3 | | Self complementarity | Max 3–4 contiguous bases | ΔG > -9 kcal/mol | | Repeat simple sequences | Filter runs of single bases | Max run length = 4 |
Primer3 operates by evaluating a target DNA sequence against a comprehensive set of user-defined constraints. The software suggests primer pairs based on several critical thermodynamic and physical parameters: Primer3—new capabilities and interfaces - PMC - NIH
To design multiplex primers using Primer 3, users simply need to:
| Parameter | Description | Example | |-----------|-------------|---------| | | Optimal length (18–27 nt) | Opt: 20 nt | | Product size range | Amplicon length (70–1000 bp) | Min 100, Max 300 bp | | Melting temperature (Tm) | Usually 50–65°C, max ΔTm ≤ 3°C | Opt: 60°C | | GC content | 40–60% recommended | Opt: 50% | | 3’-end stability | Avoid G or C in last 3–5 bases | Max poly-X = 3 | | Self complementarity | Max 3–4 contiguous bases | ΔG > -9 kcal/mol | | Repeat simple sequences | Filter runs of single bases | Max run length = 4 |