Table 1.
Exon | PCR fragment | Forward primer | Reverse primer | PCR conditions | Annealing temperaturea (°C) | Size (bp) | DHPLC temperature (°C) |
2 | 2 | GGCTGGCTGCTTTAACATTC | CTCCCCAGGCCCTATTGTAT | 2 U Taq, 40 pmol primers, 0.5 M betaine | 54 | 425 | 62 |
3 | 3 | AGGTGGTGTTGGCTCAAATC | CAACTCGACCAACCAGGTCT | 40 pmol primers | 54 | 404 | 62 |
4 | 4a | TGTGCATGCTATCCTGTCGT | GCTGGCAGAGGACTGAAGTT | 40 pmol primers | 54 | 450 | 62 |
4 | 4b | GAACTGGAGTCCCTGTGGAG | GAGGGTGGGGGTAGAAGGT | 2 U Taq, 1 mM MgCl2, 1 M betaine | 54 | 578 | 63 |
4 | 4c | GTCCCCACTCCGGTTCTG | CAGGGAGCGTAAGAGTGGAG | Standard | 54 | 442 | 63 |
4 | 4d | TGGTATATATCCCGCCTCCA | GTCCCTTCTGTTTGCTGCTC | 40 pmol primers | 54 | 436 | 57, 62 |
4 | 4e | CTTCCAACTCCACAGCCTCT | AATGGTGCTGATCAGTGCAG | 2 mM MgCl2, 0.5 M betaine | 58 | 459 | 62 |
4 | 4f | CTCGCCCTTTGTCATCTTTC | GCTGGTAGGTTTCCCATTGA | 2 mM MgCl2 | 54 | 424 | 62 |
4 | 4g | GACAGCCAAGCACAGTGAAA | GCTGAGGGTCAAGAGGACAG | Standard | 54 | 452 | 62 |
4 | 4h | CTCCTTCGTTCCAGAGCAGG | CCAGGACCAGCTCATGGGAC | Standard | 59 | 314 | 61 |
4 | 4i | AGAGAGCCACCTCTGCTCTG | ACCCCTACGCTTTCCTGTTT | Standard | 54 | 435 | 62 |
4 | 4j | AAGGTGGATGGTGATGTGGT | GAGGGGACAGCAGGTCATTA | Standard | 54 | 457 | 62 |
5 | 5 | GCAGCTCATGCCTCTAGGTC | ATCCTTGCTCGCTCACCTTA | Standard | 54 | 446 | 62 |
6 | 6 | GCAAAAGCGACCAAACTCTC | AATTCCCAACTCGACACCTG | 2 U Taq | 56 | 423 | 60 |
7 | 7 | TCCTCTGTACATCCCATCCAC | GTAGAGATGCCCGAGGGTTC | 2 mM MgCl2 | 63 | 483 | 62 |
8 | 8 | AGGCGTTGCTTTTCTGTGTT | CGCCACACACACCTTCTACA | 2 mM MgCl2 | 57 | 332 | 60 |
9 | 9 | ATGACCCTGGTGGATGGATA | GGTTCAAGCACCAGAAGAGC | Standard | 62 | 378 | 61 |
10 | 10 | TGGGCAACAGAGTGAGACTG | GCAGGCAAGGTCTTTTGAGT | Standard | 54 | 488 | 62 |
11 | 11 | CAGGTGGTTCCCTTGTCCTA | GAGCTGTTCAAGGTGGAAGG | Standard | 54 | 399 | 61 |
12 | 12 | CTTCTCCCAATTCCCTTAGCC | AAAGCCAGGGAGAAGAAAGG | 0.5 M betaine | 54 | 454 | 60 |
13 | 13 | CCCCTATATGCTCCCCTTACA | TTGCCAGGTCTTCACTTCCT | Standard | 54 | 273 | 60 |
14 | 14 | TTCCTCCAGCCTCCTTCAAT | CCCGGGACCTCTTGTCCT | 40 pmol primers | 54 | 595 | 62 |
a'Touchdown' polymerase chain reaction (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°C, and 72°C for 30 seconds. The conditions remained the same for the rest of the PCR except for the TA, 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. bp, base pairs; DHPLC, denaturing high-performance liquid chromatography.