Second-round PCR for pfdhps region containing the polymorphism | Cycling temperature | Size (bp) | To detect mutation at codon | Restriction enzyme | Fragment length (bp) wild type | Fragment length (bp) mutant |
---|---|---|---|---|---|---|
K: 5′ TGC-TAG- | 94°C for 3 min | 438 | ala | Ava II | 438 | 404 |
TGT-TAT-AGA- | Â | Â | 437 | Â | Â | Â |
TAT-AGG-ATG- | 40Â cycles of | Â | gly | Â | Â | Â |
AGC-ATC-3′ | 94°C for 1 min |  |  |  |  |  |
 | at 45°C for 45 |  | lys | FokI | 438 | 85, 320 |
K/: 5′-CTA- | sec, 72°C for 1 |  | 540 |  |  |  |
TAA-CGA-GGT-ATT-GCA-TTT-AAT-GCA-AGA-A-3′ | min, further extension 72°C for 10 min |  | glu |  |  |  |
L: 5′-ATA- | 94°C for 2 min, | 161 | ala | BstUI | 105 | 138 |
GGA-TAC-TAT- | 45°C for 2 min, |  | 581 |  |  |  |
TTG-ATA-TTG- | 72°C for 1 min |  | gly |  |  |  |
GAC-CAG- | 30Â sec (5Â cycles) | Â | Â | Â | Â | Â |
GAT-TCG-3′ L/: 5′-TAT-TAC-AAC-ATT-TTG-ATC-ATT-CGC-GCA-ACC-GG-3′ | followed by 35 cycles 94°C for 1 min; 45°C for 1 min, 72°C for 1 min 30 sec. |  |  |  |  |  |
 | Further extension at 72°C for 10 min |  |  |  |  |  |