PCR primers and restriction enzyme digests used for the detection of FLT3/TKD mutations
| . | Primers . | PCR temperature, °C . | Restriction endonuclease . | WT PCR product size, bp . | Digest, mutant, bp . |
|---|---|---|---|---|---|
| dHPLC | |||||
| gDNA | 20F: 5′-CATCACCGGTACCTCCTACTG-3′ | 63 | N/A | 278 | N/A |
| 20R: 5′-TAACGACACAACACAAAATAGCCGT-3′ | |||||
| Mutation | |||||
| D835Y | F(mm): 5′-GTGAAGATATGTGACTTTGGATTGGATCGA-3′ | 62 | ClaI | 149 | 122 |
| R: 5′-CAGTGAGTGCAGTTGTTTACCATGATAACG-3′ | |||||
| D835H | F(mm): 5′-GTGAAGATATGTGACTTTGGATTGGGTCGA-3′ | 64 | HincII | 149 | 121 |
| R: 5′-CAGTGAGTGCAGTTGTTTACCATGATAACG-3′ | |||||
| D835E | F: 5′-CATCACCGGTACCTCCTACTG-3′ | 63 | DpnII | 278 | 188 |
| R: 5′-TAACGACACAACACAAAATAGCCGT-3′ | |||||
| D835V | F: 5′-CATCACCGGTACCTCCTACTG-3′ | 64 | HincII | 217 | 187 |
| R(mm): 5′-CCTGACAACATAGTTGGAATCACTCATGTT-3′ | |||||
| Δ836 | F(mm): 5′-AAGATATGTGACTTTGGATTGGCTCGACAT-3′ | 64 | NdeI | 146 | 114 |
| R: 5′-CAGTGAGTGCAGTTGTTTACCATGATAACG-3′ | |||||
| Δ835 | F(mm): 5′-GTGAAGATATGTGACTTTGGATTGGTTCGA-3′ | 64 | BstBI | 149 | 119 |
| R: 5′-CAGTGAGTGCAGTTGTTTACCATGATAACG-3′ | |||||
| D835N | F(mm): 5′-GTGAAGATATGTGACTTTGGATTGGTTCGA-3′ | 64 | BstBI | 149 | 122 |
| R: 5′-CAGTGAGTGCAGTTGTTTACCATGATAACG-3′ |
| . | Primers . | PCR temperature, °C . | Restriction endonuclease . | WT PCR product size, bp . | Digest, mutant, bp . |
|---|---|---|---|---|---|
| dHPLC | |||||
| gDNA | 20F: 5′-CATCACCGGTACCTCCTACTG-3′ | 63 | N/A | 278 | N/A |
| 20R: 5′-TAACGACACAACACAAAATAGCCGT-3′ | |||||
| Mutation | |||||
| D835Y | F(mm): 5′-GTGAAGATATGTGACTTTGGATTGGATCGA-3′ | 62 | ClaI | 149 | 122 |
| R: 5′-CAGTGAGTGCAGTTGTTTACCATGATAACG-3′ | |||||
| D835H | F(mm): 5′-GTGAAGATATGTGACTTTGGATTGGGTCGA-3′ | 64 | HincII | 149 | 121 |
| R: 5′-CAGTGAGTGCAGTTGTTTACCATGATAACG-3′ | |||||
| D835E | F: 5′-CATCACCGGTACCTCCTACTG-3′ | 63 | DpnII | 278 | 188 |
| R: 5′-TAACGACACAACACAAAATAGCCGT-3′ | |||||
| D835V | F: 5′-CATCACCGGTACCTCCTACTG-3′ | 64 | HincII | 217 | 187 |
| R(mm): 5′-CCTGACAACATAGTTGGAATCACTCATGTT-3′ | |||||
| Δ836 | F(mm): 5′-AAGATATGTGACTTTGGATTGGCTCGACAT-3′ | 64 | NdeI | 146 | 114 |
| R: 5′-CAGTGAGTGCAGTTGTTTACCATGATAACG-3′ | |||||
| Δ835 | F(mm): 5′-GTGAAGATATGTGACTTTGGATTGGTTCGA-3′ | 64 | BstBI | 149 | 119 |
| R: 5′-CAGTGAGTGCAGTTGTTTACCATGATAACG-3′ | |||||
| D835N | F(mm): 5′-GTGAAGATATGTGACTTTGGATTGGTTCGA-3′ | 64 | BstBI | 149 | 122 |
| R: 5′-CAGTGAGTGCAGTTGTTTACCATGATAACG-3′ |
F indicates forward primer; R, reverse primer; mm, mismatch primer (mismatch underlined).