A method to amplify and express malaria parasite genes containing an intron without cDNA synthesis.

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Abstract

Background The amplification of eukaryotic genes, which contain introns for expression purposes, is very tedious and expensive. The only available lab method is through cDNA synthesis, which has a very low success rate in generating a pure gene fragment lacking any introns. Methods The current paucity of any alternative lab method directed us to devise a strategy to amplify a P. falciparum RPL12mito gene (PfRPL12mito) containing an intron through three sequential PCR steps using gDNA and primers with a minimum 15 bp 5′-overhang for the full-length protein expression. Results Sanger sequencing confirmed the absence of an intronic segment and a continuous exonic region in the amplified gene fragment. The gene was successfully expressed as recombinant protein with a 6xHis tag. Conclusions This method provides a novel and cost-effective approach to amplify intron-containing eukaryotic genes, including those from the malaria parasite, directly from genomic DNA for expression purposes.

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