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Table 2 The primer pairs, the cycling temperature and restriction enzymes used in detection of gene polymorphism on pfdhps

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

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-

  

437

   

TAT-AGG-ATG-

40 cycles of

 

gly

   

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-

30 sec (5 cycles)

     

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

     
  1. The fragment sizes of wild type and mutant are indicated.