Gene | Primer sequence | Product size (bp) | Predicted Tm (°C) |
---|---|---|---|
KRAS E2 inner | F: ATATAAACTTGTGGTAGTTGGAG | 62 | 84.1 |
R: TATCGTCAAGGCACTCTTGC | |||
KRAS E3 tailed | F: cccgggcgggccggcccCTTGGATATTCTCGACACAGCA | 85 | 92.0 |
R: cccgggcgggccggcccTCCCTCATTGCACTGTACTCCT | |||
KRAS E4 inner | F: TGGAATTCCTTTTATTGAAACATC | 56 | 75.1 |
R: TTTCAGTGTTACTTACCTGTCTTGTCT | |||
BRAF E11 inner | F: TGGGCAGATTACAGTGGGA | 68 | 79.6 |
R: GCCACTTTCCCTTGTAGACTG | |||
BRAF E15 tailed | F: gggccggcccTTCATGAAGACCTCACAGTAAA | 90 | 85.9 |
R: gggccggcccGACCCACTCCATCGAGAT |
The tailed primers for KRAS exon 3 and BRAF exon 15, as well as other primers used for the optimisation of the MSD reactions, were ‘manually’ designed using a combination of UCSC in silico PCR and MFEprimer web-based software.13 ,14
MSD, multiplex specific diagnostic; Tm, melting temperature.