Table 4.
KRAS mutations detection by AS-PCR using various mutant/wild-type DNA ratios (total 2 × 104 copies per reaction) and the several best-obtained primers for each mutation detection.
| Primers | Cq | ΔCq | ||||
|---|---|---|---|---|---|---|
| WT | 0.1% | 1% | CqWT − Cq0.1% | CqWT − Cq1% | ||
| G12A | СTTC | 37.7 ± 0.3 | 35.1 ± 0.1 | 32.0 ± 0.2 | 2.6 | 5.7 |
| С*TTC | N/A | 36.9 ± 0.2 | 32.7 ± 0.2 | 8.1 a | 12.3 a | |
| G12V | CTAT | 33.4 ± 0.1 | 33.2 ± 0.1 | 31.9 ± 0.1 | 0.2 | 1.5 |
| C*TAT | 38.7 ± 0.4 | 37.1 ± 0.2 | 33.2 ± 0.1 | 1.6 | 5.5 | |
| G12D | CGGA | 35.8 ± 0.1 | 35.1 ± 0.1 | 32.5 ± 0.1 | 0.7 | 3.3 |
| C*GGA | 41.7 ± 1.8 | 36.2 ± 0.2 | 32.9 ± 0.1 | 5.5 | 9.2 | |
| G13D | GAGA | 36.3 ± 0.1 | 34.6 ± 0.1 | 32.3 ± 0.1 | 1.7 | 4.0 |
| G*AGA | N/A | 38.5 ± 0.3 | 35.0 ± 0.1 | 6.5 a | 10.0 a | |
| GCGA | 35.0 ± 0.1 | 34.3 ± 0.1 | 31.0 ± 0.1 | 0.7 | 4.0 | |
| G*CGA | N/A | 37.0 ± 0.3 | 33.1 ± 0.1 | 8.0 a | 11.9 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 PG modification location. Boldly marked nucleotides represent mismatched nucleotides in relation to the WT DNA sequence. a If Cq(WT) = N/A to calculate ΔCq = Cq(WT) − Cq(1% or 0.1%) value Cq(WT) = 45 was used.