High prevalence of mutation in the Plasmodium falciparum dhfr and dhps genes in field isolates from Sabah, Northern Borneo

Table 1 The primer pairs, the cycling temperature and restriction enzymes used in detection of gene polymorphism on pfdhfr

Second-round PCR for *pfdhfr* region containing the polymorphism	Cycling temperature	Size (bp)	To detect mutatin at codon:	Restriction enzyme	Fragment length (bp) Wild Type	Fragment length (bp) Mutant
F: 5′-GAA-	94°C for 2 min;	326	cys	XmnI	189. 163	26, 137,
ATG-TAA-TTC-	94°C for 1 min,		59			163,
CCT-AGA-TAT-	45°C for 1 min,		arg
GGA-ATA-TT-3′	72°C for 1 min			BstNI	326	145,181
	(5 cycles)		ser
M4: 5′-TTA-			108
ATT-TCC-CAA-	continue with		thr	AluI	118, 180	299
GTA-AAA-CTA-	40 cycles,
TTA-GAG-	94°C for 1 min,		ser
CTTC-3′	45°C for 1 min,		108
	72°C for 1 min Further extension 72°C for 10 min		asp
M3: 5′-TTT-	Same cycle as	522	ala	NlaIII	53, 93, 376	146, 245
ATG-ATG-GAA-	F-M4 PCR		16
CAA-GTC-TGC-GAC-GTT- 3′			val
			asn	Tsp5091	55, 65, 120,	55, 65, 120,
F/: 5′-AAA-TTC-			51		153	218
TTG-ATA-AAC-			ile
AAC-GGA-ACC-
TTT-TA-3′
			ser	BsrI	522	190, 332
			108
			asn
			ile	Dra I	107, 171,	107, 143,
			164		245	245
			leu

ISSN: 1475-2875