Table 3.
Effect of position and type of mismatch on ligation efficiency and mismatch discrimination
| Target oligo | 5′ Arm | 3′ Arm | Ct | ΔCt (Ctx − CtD0) | Discrimination factor | ||
|---|---|---|---|---|---|---|---|
| L (nt) | Tm | L (nt) | Tm | ||||
| D0 | 20 | 59.2 | 19 | 53.1 | 19.6 ± 0.8 | na | na |
| D1 | 20 | 59.2 | 18 | 50.9 | 27.6 ± 0.5 | 8.0 | 115 |
| D2 | 20 | 59.2 | 18 | 50.4 | 28.3 ± 0.4 | 8.7 | 175 |
| D3 | 20 | 59.2 | 18 | 50.9 | 26.6 ± 0.1 | 7.0 | 64 |
| D4 | 19 | 58.4 | 19 | 53.1 | 21.7 ± 0.6 | 2.1 | 4.0 |
| D5 | 19 | 57.4 | 19 | 53.1 | 19.9 ± 0.6 | 0.3 | 1.2 |
| D6 | 19 | 57.9 | 19 | 53.1 | 26.2 ± 0.3 | 6.6 | 50 |
Lengths (L) and melting temperatures (Tms) of PLP target-complementary regions are indicated. The Ct values to quantify ligated PLPs were determined in three independent experiments. The mean values of Ct are shown along with the standard deviations to indicate reproducibility. Discrimination factor was calculated as fold-difference in the yield of ligation product, determined based on ΔCt values and amplification efficiency (E = 0.81).