Table 1. Oligonucleotide primer and probes default parameters used in qPCR automated assay design (Module 4).
| Optimal | Range | Other | |
|---|---|---|---|
| Amplicon size | – | 80–150 bp | – |
| Primers | |||
| Length | 20 nt | 18–24 nt | – |
| Melting temperature (Tm) | 60 °C | 60–63 °C | – |
| GC% | – | 50–60% | – |
| Maximum Tm difference between primers | 1.0 | – | – |
| Maximum 3′ self-complementary | 1.0 | – | – |
| Maximum poly X | 3.0 | – | – |
| 3′-end | – | No G or C | |
| Hybridization probe | |||
| Length | – | 18–30 nt | – |
| Melting temperature (Tm) | – | 65–70 °C | – |
| GC% | – | 45–65% | – |
| Maximum poly X | 3.0 | – | – |
| 5′-end | – | – | No G or C |