Amplification by PCR of PkRAP-1

Amplification of the PkRAP-1 gene was conducted by PCR using specific oligonucleotide primers PkRAP-1F: 5′-CGT TGA GCA GGA AAT GCC TAC TCC AAT C-3′ and PkRAP-1R: 5′-ATG ATA ACG TAC GCA AGT TCT CTG CTG G-3′. These primers (nucleotide positions 1782248–1782275 and 1784654–1784681) were based on the RAP-1 gene sequence of P. knowlesi strain H (GenBank Accession No. {"type":"entrez-nucleotide","attrs":{"text":"AM910995","term_id":"193810592","term_text":"AM910995"}}AM910995). The high fidelity DNA polymerase GoTaq® Long PCR Mastermix (Promega, Madison, WI, USA) was used to provide proofreading activity and efficient long DNA amplification. PCR was conducted in a total volume of 25 ml that included a final concentration of 1 × PCR mastermix, 0.4 mM of each primer and 100–500 ng of total genomic DNA. Thermal cycling profile began with an initial denaturation step at 95 °C for 2 min, followed by 35 cycles at 94 °C for 30 s and 63 °C for 2 min and 30 s, with a final extension at 72 °C for 10 min. A PCR product with an expected size of 2433 or 2434 bp was detected following electrophoresis on 1 % agarose gels.