Table 1.
Allele-specific real-time polymerase chain reaction (AS-PCR) KRAS G12A mutation detection using mutant DNA mixed with wild type (WT) DNA (total 2 × 105 copies per reaction) at various ratios (% of mutant DNA).
| Primers | Cq | ||
|---|---|---|---|
| WT | 1% | 100% | |
| CTTC | 34.25 ± 0.05 | 28.7 ± 0.1 | 21.72 ± 0.02 |
| C*TTC | N/A | 29.65 ± 0.07 | 23.8 ± 0.2 |
| CT*TC | N/A | 33.24 ± 0.01 | 26.8 ± 0.2 |
| CTT*C | N/A | 33.5 ± 0.1 | 26.67 ± 0.01 |
| C*TT*C | N/A | 36.4 ± 0.2 | 29.2 ± 0.1 |
| *C*TTC | N/A | 37.9 ± 0.1 | 32.0 ± 0.1 |
| C*T*TC | N/A | 40.9 ± 0.2 | 34.8 ± 0.1 |
| *CTT*C | N/A | N/A | 40.1 ± 0.3 |
| CT*T*C | N/A | N/A | N/A |
No template control (NTC) was undetermined in all the reactions, N/A indicates that no Cq was obtained for a typical 45-cycle reaction. Symbol “*” means phosphoryl guanidine (PG) modification location. Boldly marked nucleotides mean mismatched nucleotides in relation to the WT DNA sequence.