Table 2.
Primer Sequences and Individual Assay Conditions for All Customized SNV Genotyping Assays
| Assay target | Primer name | Primer sequence | Amplicon length, bp | Assay format | Anneal/extend temperature and time∗ | Final primer concentration, nmol/L |
|---|---|---|---|---|---|---|
| BRAF V600E (c.1799 T>A) | BRAF_600_Forward | 5′-(GGGGGG)CATGAAGACCTCACAGTAAA-3′ | Paired allele | 58°C and 1:00 | 100 | |
| BRAF_600_WT_39T | 5′-(AAATAAATAAATAAATAAATAAATAAATAAATAAATAAA)CCACTCCATCGAGATTTCA-3′ | 104 | ||||
| BRAF_V600E_Mut_6T | 5′-(GGGGGG)CCACTCCATCGAGATTTCT-3′ | 71 | ||||
|
(LNA) BRAF V600E (c.1799 T>A) |
BRAF_600_Forward | 5′-(GGGGGG)CATGAAGACCTCACAGTAAA-3′ | Paired allele | 54°C and 2:00 | 100 | |
| BRAF_600_L_WT_39T | 5′-(AAATAAATAAATAAATAAATAAATAAATAAATAAATAAA)CCACTCCATCGAGATTTC+A-3′ | 104 | ||||
| BRAF_V600E_L_Mut_6T | 5′-(GGGGGG)CCACTCCATCGAGATTTC+T-3′ | 71 | ||||
| KRAS G12D (c.35 G>A) | KRAS_12__Forward | 5′-(GCG)TGTTGGATCATATTCGTCCACAA-3′ | Paired allele | 61°C and 2:00 | 50 | |
| KRAS_12_WT | 5′-(GCG)ACTTGTGGTAGTTGGAGCAGG-3′ | 88 | ||||
| KRAS_G12D_12T | 5′-(AAATAAATAAAT)ACTTGTGGTAGTTGGAGCAGA-3′ | 100 | ||||
| KRAS G12V (c.35 G>T) | KRAS_12_Forward | 5′-(GCG)TGTTGGATCATATTCGTCCACAA-3′ | Paired allele | 63°C and 2:00 | 50 | |
| KRAS_12_WT | 5′-(GCG)ACTTGTGGTAGTTGGAGCAGG-3′ | 88 | ||||
| KRAS_G12V_Mut_100T | 5′-25(AAAT)ACTTGTGGTAGTTGGAGCAGT-3′ | 188 | ||||
| PIK3CA H1047R (c.3140 A>G) | PIK3CA_1047_Forward | 5′-(CG)CGAAAGACCCTAGCCTTAGA-3′ | Single allele | 63.5°C and 2:00 | 50 | |
| PIK3CA_1047_WT_R | 5′-(GC)TTGTCCAGCCACCATGACGT-3′ | 93 | ||||
| PIK3CA_H1047R_Mut_R | 5′-TTGTCCAGCCACCATGACGC-3′ | 93 | ||||
| TP53 R175H (c.524 G>A) | TP53_175_Forward | 5′-GGGGCCAGACCTAAGAGCAATC-3′ | Single allele | 68°C and 1:00 | 100 | |
| TP53_175_WT_R | 5′-ATGACGGAGGTTGTGAGGCG-3′ | 144 | ||||
| TP53_R175H_Mut_R | 5′-ATGACGGAGGTTGTGAGGCA-3′ | 144 | ||||
| AKT1 E17K (c.49 G>A) | AKT1_17_Reverse | 5′-GGTCTGACGGGTAGAGTGT-3′ | Single allele | 64°C and 2:00 | 50 | |
| AKT1_17_WT_F | 5′-CGCCAGGTCTTGATGTACTC-3′ | 71 | ||||
| AKT1_E17K_Mut_F | 5′-CGCCAGGTCTTGATGTACTT-3′ | 71 | ||||
| KRAS A146V (c.437 C>T) | KRAS_146_Forward | 5′-CCTAGTATAGCATAATTGAG-3′ | Single allele | 52°C and 2:00 | 50 | |
| KRAS_146_WT_R | 5′-CCTTTTATTGAAACATCGGC-3′ | 132 | ||||
| KRAS_A146V_Mut_R | 5′-CCTTTTATTGAAACATCGGT-3′ | 132 | ||||
| TP53 E285K (c.853 G>A) | TP53_285_Forward | 5′-CGCTTAGTGCTCCCTGGGGG-3′ | Single allele | 64°C and 1:30 | 50 | |
| TP53_285_WT_R | 5′-TGGGAGAGACCGGCCCATAG-3′ | 84 | ||||
| TP53_E285K_Mut_R | 5′-TGGGAGAGACCGGCCCATAA-3′ | 84 |
Tail sequences are in parenthesis, mismatch bases are underlined, and +N locked nucleic acids are boldfaced.
SNV, single-nucleotide variant.
∗
Time is given in minutes:seconds.