Table 3.
AS-PCR KRAS mutation detection using WT DNA (total 2 × 104 copies per reaction) and 1% mutant DNA in the background of WT DNA.
| Primers | Cq | ΔCq | Primers | Cq | ΔCq | ||||
|---|---|---|---|---|---|---|---|---|---|
| WT | 1% | CqWT − Cq1% | WT | 1% | CqWT − Cq1% | ||||
| G12A | CTGC | 33.0 ± 0.5 | 31.5 ± 0.1 | 1.5 | G12V | CTGT | 26.2 ± 0.1 | 26.0 ± 0.1 | 0.2 |
| С*TGC | 38.4 ± 0.4 | 32.6 ± 0.1 | 5.8 | C*TGT | 30.7 ± 0.1 | 29.7 ± 0.1 | 1.0 | ||
| CTTC | 37.7 ± 0.3 | 32.0 ± 0.2 | 5.7 | CTAT | 33.4 ± 0.1 | 31.9 ± 0.1 | 1.5 | ||
| С*TTC | N/A | 32.7 ± 0.2 | 12.3 a | C*TAT | 38.7 ± 0.3 | 33.2 ± 0.1 | 5.5 | ||
| CAGC | 37.7 ± 0.2 | 33.0 ± 0.1 | 4.7 | CTTT | 35.3 ± 0.1 | 34.2 ± 0.1 | 1.1 | ||
| C*AGC | 38.7 ± 0.3 | 32.5 ± 0.1 | 6.2 | C*TTT | 41.8 ± 1.1 | 37.9 ± 0.3 | 3.9 | ||
| G13D | GTGA | 29.0 ± 0.1 | 28.1 ± 0.1 | 0.9 | CGGT | 28.0 ± 0.1 | 27.4 ± 0.1 | 0.6 | |
| G*TGA | 33.4 ± 0.1 | 32.3 ± 0.1 | 1.1 | C*GGT | 28.5 ± 0.1 | 27.6 ± 0.1 | 0.9 | ||
| GAGA | 36.3 ± 0.2 | 32.3 ± 0.1 | 4.0 | CCGT | 31.7 ± 0.1 | 30.0 ± 0.1 | 1.7 | ||
| G*AGA | N/A | 35.0 ± 0.1 | 10.0 a | C*CGT | 33.9 ± 0.2 | 30.9 ± 0.1 | 3.0 | ||
| GCGA | 35.0 ± 0.1 | 31.0 ± 0.1 | 4.0 | G12D | CTGA | 27.7 ± 0.1 | 27.0 ± 0.1 | 0.7 | |
| G*CGA | N/A | 33.1 ± 0.1 | 11.9 a | C*TGA | 29.2 ± 0.1 | 28.7 ± 0.1 | 0.5 | ||
| GGGA | 38.0 ± 0.4 | 32.8 ± 0.1 | 5.2 | CGGA | 35.8 ± 0.1 | 32.50 ± 0.05 | 3.3 | ||
| G*GGA | N/A | 36.4 ± 0.2 | 8.6 a | C*GGA | 41.7 ± 1.8 | 32.9 ± 0.1 | 8.8 | ||
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. SNP G/C (G12A), G/T (G12V), G/A (G12D, G13D); 3′-end mismatch site with mutated DNA C/C, Pyr/Pyr (G12A); C/T, Pyr/Pyr (G12V); C/A, Pyr/Pur (G12D, G13D). a If Cq(WT) = N/A to calculate ΔCq = Cq(WT) − Cq(1%) value Cq(WT) = 45 has been used.