Primer sequences, PCR conditions, and sensitivity of the ASO-PCR assays used in this study
Mutant . | Base exchange . | Frequency, %* . | Primer names . | Sequence (5′ → 3′) . | Annealing temperature, °C . | No. cycles . | Product length, bp . | Extension time, s . | Detection limit, mutant/wild type . |
|---|---|---|---|---|---|---|---|---|---|
| Q252H | G-C | 5.1 | 252H C3R | TCGTACACCTCCCCGCAG | 60.0 | 45 | 789 | 60 | ≤ 1/100 000 |
| Bcr 2687F | CGCTGACCATCAATAAGG | ||||||||
| Y253F | A-T | 3.4 | 253F R | CTCGTACACCTCCCCGA | 60.0 | 45 | 790 | 60 | ≤ 1/100 000 |
| Bcr2687F | CGCTGACCATCAATAAGG | ||||||||
| Y253H | T-C | 6.8 | 253H 16R | CGTACACCTCCCCGTG | 60.4 | 45 | 805 | 60 | ≤ 1/1000 |
| Bcr 2671F | ACTCCAGACTGTCCACAGCAT | ||||||||
| E255K | G-A | 19.2 | 255K T3R | CACGCCCTCGTACACTTT | 60.0 | 45 | 798 | 60 | ≤ 1/100 000 |
| Bcr2687F | CGCTGACCATCAATAAGG | ||||||||
| E255V | A-T | 2.3 | 255V T3R | ACACGCCCTCGTACATCA | 60.0 | 45 | 797 | 60 | ≤ 1/100 000 |
| Bcr2687F | CGCTGACCATCAATAAGG | ||||||||
| T315I | C-T | 20.9 | 315 R | CGTAGGTCATGAACTCAA | 60.0 | 50 | 994 | 80 | ≤ 1/100 000 |
| Bcr2671F | ACTCCAGACTGTCCACAGCAT | ||||||||
| M351T | T-C | 15.3 | 351 G3R | TCTTCTCCAGGTACTGCG | 60.0 | 50 | 1085 | 90 | ≤ 1/100 000 |
| Bcr2687F | CGCTGACCATCAATAAGG | ||||||||
| F359V | T-G | 4.0 | 359 R | AAGATCTCTGTGGATGAC | 60.0 | 50 | 1107 | 90 | ≤ 1/100 000 |
| Bcr2687F | CGCTGACCATCAATAAGG |
Mutant . | Base exchange . | Frequency, %* . | Primer names . | Sequence (5′ → 3′) . | Annealing temperature, °C . | No. cycles . | Product length, bp . | Extension time, s . | Detection limit, mutant/wild type . |
|---|---|---|---|---|---|---|---|---|---|
| Q252H | G-C | 5.1 | 252H C3R | TCGTACACCTCCCCGCAG | 60.0 | 45 | 789 | 60 | ≤ 1/100 000 |
| Bcr 2687F | CGCTGACCATCAATAAGG | ||||||||
| Y253F | A-T | 3.4 | 253F R | CTCGTACACCTCCCCGA | 60.0 | 45 | 790 | 60 | ≤ 1/100 000 |
| Bcr2687F | CGCTGACCATCAATAAGG | ||||||||
| Y253H | T-C | 6.8 | 253H 16R | CGTACACCTCCCCGTG | 60.4 | 45 | 805 | 60 | ≤ 1/1000 |
| Bcr 2671F | ACTCCAGACTGTCCACAGCAT | ||||||||
| E255K | G-A | 19.2 | 255K T3R | CACGCCCTCGTACACTTT | 60.0 | 45 | 798 | 60 | ≤ 1/100 000 |
| Bcr2687F | CGCTGACCATCAATAAGG | ||||||||
| E255V | A-T | 2.3 | 255V T3R | ACACGCCCTCGTACATCA | 60.0 | 45 | 797 | 60 | ≤ 1/100 000 |
| Bcr2687F | CGCTGACCATCAATAAGG | ||||||||
| T315I | C-T | 20.9 | 315 R | CGTAGGTCATGAACTCAA | 60.0 | 50 | 994 | 80 | ≤ 1/100 000 |
| Bcr2671F | ACTCCAGACTGTCCACAGCAT | ||||||||
| M351T | T-C | 15.3 | 351 G3R | TCTTCTCCAGGTACTGCG | 60.0 | 50 | 1085 | 90 | ≤ 1/100 000 |
| Bcr2687F | CGCTGACCATCAATAAGG | ||||||||
| F359V | T-G | 4.0 | 359 R | AAGATCTCTGTGGATGAC | 60.0 | 50 | 1107 | 90 | ≤ 1/100 000 |
| Bcr2687F | CGCTGACCATCAATAAGG |
The last nucleotide of the reverse primer matches the mutated base. Additional mismatches versus wild type are in bold and underlined.
N = 177.9