Table 2.
Multiplex TP53 Primer Sequences from Fredriksson et al.37
| Exon | Sequence | Size (bp) | Tm°C | Tc°C | Ta°C |
|---|---|---|---|---|---|
| Multiplex primers | |||||
| 2-3 | Forward-5′-ATGCTGGATCCCCACTTTTC-3′⁎ | 350 | |||
| Reverse-5′-GACCAGGTCCTCAGCCC-3′⁎ | |||||
| 4 | Forward-5′-GACAAGGGTTGGGCTGG-3′⁎ | 486 | |||
| Reverse-5′-CCAAAGGGTGAAGAGGAATC-3′⁎ | |||||
| 5-6 | Forward-5′-TCTTTGCTGCCGTCTTCC-3′⁎ | 517 | |||
| Reverse-5′-AGGGCCACTGACAACCAC-3′⁎ | |||||
| 7 | Forward-5′-TGCTTGCCACAGGTCTCC-3′⁎ | 235 | |||
| Reverse-5′-GTCAGAGGCAAGCAGAGGC-3′⁎ | |||||
| 8-9 | Forward-5′-GGACAGGTAGGACCTGATTTCC-3′⁎ | 441 | |||
| Reverse-5′-AAACAGTCAAGAAGAAAACGGC-3′⁎ | |||||
| 10 | Forward-5′-AACTTGAACCATCTTTTAACTCAGG-3′⁎ | 243 | |||
| Reverse-5′-GGAATCCTATGGCTTTCCAAC-3′⁎ | |||||
| 11 | Forward-5′-AGGGGCACAGACCCTCTC-3′⁎ | 222 | |||
| Reverse-5′-AGACCCAAAACCCAAAATGG-3′⁎ | |||||
| Nested primers | |||||
| 2 | Forward-5′-GCAGCCAGACTGCCTTCCG-3′ | 134 | 90.6 | 89.6 | 57 |
| Reverse-5′-GTGGGCCTGCCCTTCCAAT-3′ | |||||
| 3 | Forward-5′-gtaaaacgacggccagtTGGGACTGACTTTCTGCT-3′† | 108 | 86.8 | 85.9 | 58 |
| Reverse-5′-tcccgcgaaattaatacgacGCCCAACCCTTGTCCTTA-3′† | |||||
| 4a | Forward-5′-GTCCTCTGACTGCTCTTTTCACCC-3′ | 181 | 90.8 | 89.8 | 60 |
| Reverse-5′-GGTGTAGGAGCTGCTGGTGC-3′ | |||||
| 4b | Forward-5′-CCCGTGGCCCCTGCACC-3′ | 186 | 88.8‡ | 87.8 | 70 |
| Reverse-5′-AGCCAGCCCCTCAGGGCAA-3′ | |||||
| 5a | Forward-5′-TGTGCCCTGACTTTCAACTCTGTCTC-3′ | 120 | 88.8 | 87.7 | 68 |
| Reverse-5′-GGGTGTGGAATCAACCCACAGC-3′ | |||||
| 5b | Forward-5′-TTCCACACCCCCGCCCG-3′ | 148 | 94.5 | 93.5 | 63 |
| Reverse-5′-GCCCTGTCGTCTCTCCAGCC-3′ | |||||
| 6a | Forward-5′-GCCTCTGATTCCTCACTGATTG-3′ | 129 | 86.9 | 86.0 | 57 |
| Reverse-5′-TAGGGCACCACCACACTATG-3′ | |||||
| 6b | Forward-5′-TGCGTGTGGAGTATTTGGATGAC-3′ | 105 | 88.5 | 87.5 | 57 |
| Reverse-5′-CCCTCCTCCCAGAGACCC-3′ | |||||
| 7 | Forward-5′-CCAAGGCGCACTGGCCTCA-3′ | 185 | 91.0 | 90.0 | 57 |
| Reverse-5′-GCCAGTGTGCAGGGTGGCAA-3′ | |||||
| 8a | Forward-5′-TGCCTCTTGCTTCTCTTTTC-3′† | 128 | 88.8 | 88.0 | 57 |
| Reverse-5′-CTTTCTTGCGGAGATTCTCTTC-3′† | |||||
| 8b | Forward-5′-GAACAGCTTTGAGGTGCGTGTTT-3′ | 155 | 91.4 | 90.5 | 57 |
| Reverse-5′-TGGTCTCCTCCACCGCTTC-3′ | |||||
| 9 | Forward-5′-GGTGCAGTTATGCCTCAGAT-3′ | 176 | 87.2 | 86.1 | 57 |
| Reverse-5′-GTTAGACTGGAAACTTTCCACTTGATA-3′ | |||||
| 10 | Forward-5′-ATATACTTACTTCTCCCCCTCCTCTGTTGC-3′ | 172 | 92.8 | 91.8 | 70 |
| Reverse-5′-TAGGGCCAGGAAGGGGCTGA-3′ | |||||
| 11 | Forward-5′-CTCACTCATGTGATGTCATCTCT-3′ | 165 | 88.1 | 87.2 | 57 |
| Reverse-5′-GGGAGGCTGTCAGTGGG-3′ |
Nested primers were used to amplify the length of each TP53 exon via both conventional and COLD-PCR. Nested Exon 3 primers were modified to include M13 and Tag1 linker sequences (lower case font) in order to increase the product length and allow sequence analysis. The annealing (Ta), amplicon melting temperature (Tm), and critical denaturation temperature (Tc) of COLD-PCR are presented for PCR reactions performed with the Phusion high-fidelity polymerase system and a 1X concentration of LCGreen+ dye (Idaho Technologies Inc.). When necessary (Exons 4, 5, 6, and 8), two amplicons were analyzed in order to screen the entire length of the exon; primer sets are differentiated by “a” and “b” labels.
Fredriksson et al.37
Bastien et al.23
DMSO was added at 5% to lower the amplicon melting temperature.