Gene Fragment | Primer name | Â | Primer Sequence | Codons | PCR Cycling conditions |
---|---|---|---|---|---|
Fragment 3 | |||||
Primary FR3 | MDRF3N1 | F | 5'-GCATTTTATAATATGCATACTG-3' | 1034, 1042 | 94°C 3 min/[94°C 30 s-56°C 60 s 65°C 50 s] × 30 cycles 65°C 5 min/15°C 5 min |
 | MDRF3R1 | R | 5'-GGATTTCATAAAGTCATCAAC-3' |  |  |
Nested FR3 | MDRF3N2 | F | 5'-GGTTTAGAAGATTATTTCTGTA-3' | Â | Â |
 | MDRF3R1 | R | 5'-GCATTTTATAATATGCATACTG-3' |  |  |
Fragment 4 | |||||
Primary FR4 | MDRF4N1 | F | 5'-CAAACCAATCTGGATCTGCAG-3' | 1246 | 94°C 3 min/[94°C 30 s-55°C 60 s-65°C 40 s] × 30 cycles 65°C 5 min/15°C 5 min |
 | MDRF4R1 | R | 5'-CAATGTTGCATCTTCTCTTCC-3' |  |  |
Nested FR4 | MDRF4N2 | F | 5'-GATCTGCAGAAGATTATACTG-3' | Â | Â |
 | MDRF4R1 | R | 5'-CAATGTTGCATCTTCTCTTCC-3' |  |  |
Sequencing | |||||
FR3 | MDRF3N2 | F | 5'-GGTTTAGAAGATTATTTCTGTA-3' |  | 96°C 1 min [96°C 30 s-50°C-60°C 4 min] × 26 cycles |
 | MDRF3R1 | R | 5'-GCATTTTATAATATGCATACTG-3' |  |  |
FR4 | MDRF4N2 | F | 5'-GATCTGCAGAAGATTATACTG-3' | Â | Â |
 | MDRF4R1 | R | 5'-CAATGTTGCATCTTCTCTTCC-3' |  |  |