Table 1.
Exon | PCR fragment | Forward primer | Reverse primer | Annealing temperature (°C)a | Size (bp) | DHPLC temperature (°C) |
1 | 1 | GCAAGCGAGTAGAGAAGTGGA | AACTGCCGCTGAGCACTG | 54 | 218 | 65 |
2 | 2 | ATGGCCTTGGCTTTTCCTAA | GGCCCTGCCAGACATATTTA | 54 | 391 | 57 |
3 | 3 | TGGAACCAACTTCCCATCTC | TTCCCACTAATGCCTCCCTA | 54 | 341 | 58 |
4 | 4 | CTCTTCCCATTGCACACCTT | CACCTGGCCTTCCTCTATCTC | 54 | 371 | 52, 57 |
5 | 5 | TCTGATGAGCTCCAAGAACA | TGACATGGAAGGATTTTGAAG | 51 | 344 | 58 |
6 | 6 | AAGGGAATGCCTCCTTCCTA | CCAAACTAACCCTGGCAATC | 54 | 314 | 59 |
7 | 7 | CAAATTGCTCATCTGCCTG | TGAGGCACCGTTTAACAAGA | 54 | 397 | 59 |
8 | 8 | TGGTAAGGAAGGGACCAGAA | TGTGGCCATAGACACTCCAA | 57 | 390 | 60 |
9 | 9 | TCGTTATTTTGTGGGGGAAA | ACAGGGGAGAGGCATATCAA | 54 | 447 | 58 |
10 | 10a | TTGGTGCTTTGGTCTGTGTC | ATACCCCTCCTCCAAAACCA | 54 | 409 | 59 |
10 | 10b | CAGGAGACAGGTCAGTAGTCACA | AGGTTTGGCACAAGACTCCA | 51 | 374 | 57 |
10 | 10c | TGATCTTGTGTAAGGGTTTGGTT | GCAATCTCGACTCACTGCAA | 51 | 375 | 60 |
10 | 10d | GATAGCCTGAGGTGGGAGAA | TCTGCAAGTGGGACTTTCCT | 54 | 319 | 60 |
a'Touchdown' PCR amplification conditions were as follows: denaturation at 94°C for 10 minutes, followed by two cycles of 94°C for 30 seconds, 30 seconds at the fragment annealing temperature (TA) + 6°, and 72°C for 30 seconds. The conditions remained the same for the rest of the PCR except for the annealing temperature, which consisted of two cycles at (TA + 4°C), then two cycles at (TA + 2°C) and, finally, 35 cycles at the TA. A final extension step was conducted at 72°C for 7 minutes.