TABLE 1.
PCR oligonucleotide primer sequences and thermocycling conditions for target sequences in pfcrt, pfdhps, pfdhfr, and pfmdr1g
| Gene fragment | PCR primer sequence | Thermocycling conditionsf |
|---|---|---|
| pfcrta | 5′-TAATACGACTCACTATAGGGCCGTTA-3′ | 35 cycles of 95°C for 30 s, 56°C for 30 s, 60°C for 1 min |
| 5′-ATTAACCCTCACTAAAGGGACGGATG-3′ | ||
| pfdhfrb | 5′-TAACTACACATTTAGAGGTCTA-3′ | 35 cycles of 95°C for 30 s, 56°C for 30 s, 72°C for 1 min |
| 5′-GTTGTATTGTTACTAGTATATAC-3′ | ||
| pfdhpsc | 5′-AATGATAAATGAAGGTGCTAGT-3′ | 35 cycles of 95°C for 30 s, 56°C for 30 s, 60°C for 1 min |
| 5′-ATGTAATTTTTGTTGTGTATTTA-3′ | ||
| pfmdr1 3′ fragmentd | 5′-TGTATGTGCTGTATTATCAG-3′ | 32 cycles of 94°C for 30 s, 56°C for 30 s, 72°C for 30 s |
| 5′-CTTATTACATATGACACCACA-3′ | ||
| pfmdr1 5′ fragmente | 5′-TAGAAGATTATTTCTGTAATTTG-3′ | 40 cycles of 94°C for 30 s, 56°C for 30 s, 72°C for 1 min |
| 5′-CAATGTTGCATCTTCTCTTCCA-3′ |
a
pfcrt for SNPs at codons 72 to 76.
b
pfdhfr fragment for SNPs at codons 51, 59, 108, and 164.
c
pfdhps gene fragment for SNPs at codons 540, 581, and 613.
d
pfmdr1 3′ fragment for SNPs at codons 86 and 184.
e
pfmdr1 5′ fragment for SNPs at codons 1034, 1042, and 1246.
f
PCR programs were preceded by an initial denaturation step at 95°C for 2 min.
g
Conditions for pfdhfr and pfcrt were optimized to obviate the need for nested reactions.