Table 1.
Sequences of probes and primers designed for the mutation detection assays.
| Assay | Oligonucleotide names | oligonucleotide sequences | Modifications | Tm °C |
|---|---|---|---|---|
| 69/70 deletion | Del 69/70 FW | GACTTGTTCTTACCTTTCTTTTCC | 60.3 | |
| Del 69/70 RV | CATCATTAAATGGTAGGACAGGG | 60.9 | ||
| Probe 69/70 | TCCATGCTATACA(T)GTCTCTGGGACCAAT | 3´C3 T-BHQ1 5´FAM |
69.1 | |
| Probe Del 6970 | GTTCCATGCTATC(T)CTGGGACCAATGGT | 3´C3 T-BHQ2 5´Cal Fluor Red 610 |
70.1 | |
| K417N mutation |
K417N RV | AATTACCACCAACCTTAGAATCAAG | 60.9 | |
| 417K FW | CTCCAGGGCAAACTGGAAA+G | G LNA | 65 | |
| 417N FW | GCTCCAGGGCAAACTGGAAA+T | T LNA | 66 | |
| Probe K417N | CCAGATGATTTTACAGGCTGCGTTATAG | 3´BHQ3 5´Quasar 670 |
67 | |
| E484K/N501Y mutations (duplex-assay) |
484/501 FW | ATCTATCAGGCCGGTAGCAC | 60.5 | |
| 484/501 RV | GTACTACTACTCTGTATGGTTGG | 60.9 | ||
| Probe 484E | CTTGTAATGGTGTTGAAGGTTTTAATTG | 3´BHQ1 5´FAM |
62.7 | |
| Probe 484K | CTTGTAATGGTGTTAAAGGTTTTAATTG | 3´BHQ2 5´Cal Fluor Red 610 |
61.5 | |
| Probe 501N | TCCAACCCACT+AATGGTGTTGG | 3´BHQ1 A LNA 5´HEX |
66 | |
| Probe 501Y | TCCAACCCACT+TATGGTGTTGG | 3´BHQ3 T LNA 5´Quasar 670 |
66 |
The letter “T” in parentheses denotes the position of the internal quencher. The symbol “+” indicates that the next base is a modified base (LNA).