Mutation | Sense primer (nucleotide position) | Antisense primer (table1) | Product size (bp) | Annealing temp (°C) | MgCl2 conc (mmol/l) | Restriction enzyme (fragment sizes (bp)) |
D32G | 5′ACTCTCTGTCTCCCACCTCACGCAGCGGTT3′ (67-962-150) | SFA2(−) | 288 | 64 | 1.5 | −HincII (288) |
G122R | 5′GCCAGGCCTCTCTTCCCGCCCAGGCATCCT3′ (503-532†) | SFA4(−) | 243 | 62 | 1.0 | +DdeI (29+133+81) |
D235N | 5′TTCGTCCCCAACACCCCGGCAGCCCGAGTC3′ (536-565‡) | SFA6(−) | 201 | 64 | 1.5 | −TaqI (201) |
D273N | 5′CCTGAACGACACACTGGTGATCTTCACGAC3′ (170-199§) | SFA7(−) | 274 | 64 | 1.5 | −BsiEI (274) |
Y374H | 5′CAGCCAGAGCCACCACGAGGTCACCATGGC3′ (1532-1561§) | SFA8a(−) | 229 | 64 | 1.5 | +Sau96I (28+111+48+42) |
↵2-150 † ‡ §Positions of primers are numbered according to Genbank database entry u60108, u60109, u60110, and u60111, respectively.7