Table 5.
ASB-PCR KRAS mutations detection of various mutant/wild-type DNA ratios (total 2 × 104 or 2 × 105 copies per reaction).
| Primers | Cq | ΔCq | |||||
|---|---|---|---|---|---|---|---|
| CqWT − Cq1% | |||||||
| 2 × 104 Copies | 2 × 104 | 2 × 105 | |||||
| 1% | 0.1% | 0.01% | WT | Copies | |||
| G12A | No blocker CTGG | 24.6 ± 0.2 | 24.8 ± 0.2 | 25.0 ± 0.3 | 25.0 ± 0.2 | 0.4 | - |
| Blocker C*T*G*G | N/A | N/A | N/A | N/A | - | - | |
| No blocker CTTC | 31.5 ± 0.1 | 33.9 ± 0.1 | 34.2 ± 0.2 | 36.7 ± 0.2 | 5.2 | 5.7 | |
| CTTC/C*T*G*G = 1/0.1 | 33.5 ± 0.1 | 38.0 ± 0.3 | N/A | N/A | 11.5 a | 10.7 | |
| C*TTC/C*T*G*G = 1/0.25 | 32.5 ± 0.1 | 36.3 ± 0.3 | 38.2 ± 0.4 | N/A | 12.5 a | 14.2 | |
| C*TGC/C*T*G*G = 1/0.25 | 33.1 ± 0.1 | 37.5 ± 0.2 | 40.1 ± 0.5 | 42.4 ± 1.5 | 9.3 | 9.2 | |
| G12V | No blocker CTAT | 30.6 ± 0.1 | 30.8 ± 0.2 | 31.4 ± 0.2 | 31.6 ± 0.2 | 1.0 | - |
| C*TAT /C*T*G*G = 1/0.1 | 36.0 ± 0.2 | 37.5 ± 0.4 | 40.3 ± 1.0 | N/A | 9.0 a | 8.8 | |
| G12D | No blocker CGGA | 33.2 ± 0.1 | 36.0 ± 0.3 | 37.0 ± 0.5 | 37.1 | 3.8 | - |
| C*GGA/C*T*G*G = 1/0.1 | 33.6 ± 0.1 | 37.1 ± 0.6 | 39.2 ± 0.9 | N/A | 11.4 a | 9.8 | |
| G13D | No blocker GTGG | 23.5 ± 0.1 | 24.0 ± 0.2 | 24.1 ± 0.1 | 24.2 ± 0.3 | 0.7 | - |
| Blocker G*T*G*G | N/A | N/A | N/A | N/A | - | - | |
| No blocker GAGA | 31.1 ± 0.1 | 33.1 ± 0.2 | 33.6 ± 0.3 | 35.2 ± 0.2 | 4.1 | - | |
| GAGA/G*T*G*G = 1/0.1 | 32.7 ± 0.1 | 34.8 ± 0.2 | N/A | N/A | 12.3 a | 8.8 | |
| G*AGA/G*T*G*G = 1/0.1 | 35.3 ± 0.4 | 39.2 ± 0.8 | 41.0 ± 1.3 | N/A | 9.7 a | 10.9 | |
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. Real-time PCR assay was done using a constant 450 nM AS-primers concentration and several blocker primers ratios. a If Cq(WT) = N/A to calculate ΔCq = Cq(WT) − Cq(1%) value Cq(WT) = 45 was used.