Table 1.
Primers and Conditions Used for Amplification of Exons
| Primer | Sequence | Size (nt) | Taa (°C) | Bufferb |
| 2521 | TGGAGAAACTTCCTCTTTTTC | |||
| 2438 | GACTGGAGCTCAGGAGAAAGC | 92 | 65–55 | 1 |
| Exon1/f | CACGGAAGACAGCAGACAAA | |||
| Exon1/r | GTAGNGCACCCCAGTTTCCC | 604 | 65–50 | 1 |
| Exon2/f | CTCTAGGTGGGTGAGGCAGT | |||
| Exon2/r | CTAGGGATGTGGAGGGACTG | 134 | 70–55 | 2 |
| Exon3/f | TTTTGGGCTACTCAGTTATGCTA | |||
| Exon3/r | AGCACAGGCAGACAGATGTG | 273 | 70–55 | 2 |
| Exon4/f | AATTCTATGCCATTTATTTCCC | |||
| Exon4/r | ACATTGACTAGTTTGCAGCTC | 297 | 65–50 | 1 |
| Exon5/f | CGCTCACACTGAGACCTGAC | |||
| Exon5/r | ATTAGCACAGGGACACCAGG | 202 | 65–50 | 1 |
| Exon6/f | TAGAAACGAGGCCTGGAGAA | |||
| Exon6/r | ATGGGTGCCTATGTGAGAGG | 353 | 65–50 | 1 |
| Exon7/f | CTCACCCATTGCTTCTGAC | |||
| Exon7/r | AAGCATTATTGGAAGAGGACAA | 262 | 70–55 | 2 |
a
“Touchdown” PCR was used: the annealing temperature Ta was decreased by 1°C/cycle for the first 10 or 15 cycles, followed by 25 cycles at the final annealing temperature. Initial and final annealing temperatures are shown.
b
1 = Standard PE Biosystems/Cetus buffer with 1.5 mM Mg++; 2 = Kogan buffer: 16.6 mM ammonium sulfate, 67 mM Tris-Cl- (pH 8.8 at 25°C), 6.7 mM MgCl2, 10 mM β-mercaptoethanol, 6.7 μM EDTA, 170 μg BSA per ml, and 10% dimethylsulfoxide (Kogan et al. 1987).