Primer sequences used for the PDM, MSD and SSD reactions
| Gene | Primer sequence | Product size (bp) | Predicted Tm (°C) |
|---|---|---|---|
| KRAS E2 outer | F: TGAATATAAACTTGTGGTAGTTGG | 174 | NA |
| R: GCTGTATCGTCAAGGCACTCT | |||
| KRAS E2 inner | F: ATATAAACTTGTGGTAGTTGGAG | 62 | 84.1 |
| R: TATCGTCAAGGCACTCTTGC | |||
| KRAS E3 outer | F: CCAGACTGTGTTTCTCCCTTC | 152 | NA |
| R: AAAGAAAGCCCTCCCCAGT | |||
| KRAS E3* inner | F: TGTGTTTCTCCCTTCTCAGGA | 145 | 86.3 |
| R: AAGAAAGCCCTCCCCAGT | |||
| KRAS E4 outer | F: AGACACAAAACAGGCTCAGGA | 160 | NA |
| R: TTGAGAGAAAAACTGATATATTAAATGAC | |||
| BRAF E15 outer | F: ATCTACTGTTTTCCTTTACTTACTACAC | 205 | NA |
| R: CAGCATCTCAGGGCCAA | |||
| PIK3CA E1 outer | F: CACGACCATCATCAGGTGAA | 168 | NA |
| R: GGAGGGGGTATTTTCTTGCT | |||
| PIK3CA E9 outer | F: CTGTGAATCCAGAGGGGAAA | 197 | NA |
| R: GCACTTACCTGTGACTCCATAGAA | |||
| PIK3CA E20 outer | F: TGAGCAAGAGGCTTTGGAGT | 201 | NA |
| R: CCTATGCAATCGGTCTTTGC | |||
| PIK3CA E20 inner | F: GCAAGAGGCTTTGGAGTATTTC | 115 | 81.9 |
| R: TTTTCAGTTCAATGCATGCTG | |||
| PTEN E3 outer | F: TCATTTTTGTTAATGGTGGCTTT | 182 | NA |
| R: ACTCTACCTCACTCTAACAAGCAGA | |||
| PTEN E3 inner | F: GGCTTTTTGTTTGTTTGTTTTG | 158 | 78.4 |
| R: CCTCACTCTAACAAGCAGATAACTTTC | |||
| PTEN E5A outer | F: GGTTATCTTTTTACCACAGTTGCAC | 118 | NA |
| R: GATTGTCATCTTCACTTAGCCATT | |||
| PTEN E7 outer | F: GTTCCCTCAGCCGTTACCT | 191 | NA |
| R: CACCTGCAGATCTAATAGAAAACAA | |||
| NRAS E2 outer | F: GGTTTCCAACAGGTTCTTGC | 191 | NA |
| R: TCCGACAAGTGAGAGACAGG | |||
| NRAS E2 inner | F: TACAAACTGGTGGTGGTTGG | 115 | 86.6 |
| R: CACTGGGCCTCACCTCTATG |
*These primers were tailed and used for the MSD reactions. The outer and inner primers were designed using Primer3 as previously described.6 These primers have also been used in previous studies.6 The KRAS exon 3 and BRAF exon 15 tailed primers were used for the MSD reactions.
MSD, multiplex specific diagnostic; NA, not applicable; PDM, prediagnostic multiplex; SSD, single specific diagnostic.