Table 1.
General Guidelines for Handling 3′ End Primer-Template Mismatches during Real-Time PCR Development
| Recommendations per Experimental Setup (for detection/quantification of nucleic acids) | ||||
|---|---|---|---|---|
| Mismatch (single) | Position (primer) | Standard real-time PCR (Taq DNA polymerase-based) | Real-time RT-PCR using specific reverse primer (Taq DNA polymerase) | rTth DNA polymerase-based real-time PCR using specific reverse primer | 
| A–A/A–G/G–A/G–G/C–C | 3′ terminal | Avoid | Avoid in FWD, | Acceptable in FWD, | 
| (both primers) | Acceptable in REV | Avoid in REV | ||
| 3′ penultimate | Avoid | Avoid in FWD, | Acceptable in FWD, | |
| (both primers) | Acceptable in REV | Avoid in REV | ||
| 3′ positions 3-5 | Acceptable | Avoid in FWD, | Acceptable in FWD, | |
| (both primers) | Acceptable in REV | Avoid in REV | ||
| T–T/T–C/C–T | 3′ terminal | Avoid | Avoid in FWD, | Acceptable in FWD, | 
| (both primers) | Acceptable in REV | Avoid in REV | ||
| 3′ penultimate | Acceptable | Avoid in FWD, | Acceptable in FWD, | |
| (both primers) | Acceptable in REV | Avoid in REV | ||
| 3′ positions 3-5 | Acceptable | Acceptable | Acceptable in FWD, | |
| (both primers) | (both primers) | Avoid in REV | ||
| C–A/A–C/G–T/T–G | 3′ terminal | Acceptable | Avoid in FWD, | Acceptable in FWD, | 
| (both primers) | Acceptable in REV | Avoid in REV | ||
| 3′ penultimate | Acceptable | Acceptable | Acceptable in FWD, | |
| (both primers) | (both primers) | Avoid in REV | ||
| 3′ positions 3-5 | Acceptable | Acceptable | Acceptable in FWD, | |
| (both primers) | (both primers) | Avoid in REV | ||
| Best discriminating properties | - A–A/A–G/G–A/G–G/C–C at 3′ terminal position of the primer | |||
| - A–A/A–G/G–A/G–G/C–C at 3′ penultimate position of the primer | ||||
| - T–T/T–C/C–T at 3′ terminal position of the primer | ||||
| (primer choice will depend on the type of experimental setup used) | ||||
Mismatches are depicted as primer–template mismatches. Mismatches were designated as acceptable (for general applications) when their effect was generally <2,0 Ct. FWD = forward primer, REV = reverse primer.