Fig. 5

Divergent primers and Sanger sequencing validate circRNA splicing in P. falciparum. (A) The apoptosis-related protein (ARP) encodes a predicted circRNA, termed ARP_circRNA, consisting of ARP exon-3 and exon-4 sequence. (B) To validate the non-canonical exon-4 donor (GT)/exon-3 acceptor (AG) splice junction in ARP_circRNA, we designed a divergent PCR primer pair. The primer pair is considered to be divergent, rather than convergent, because the reverse primer binds upstream of the forward primer. (C) PCR using divergent primers amplified a product of the expected size (161 bp, indicated with an arrow) when the template was cDNA from either time course, but not water or gDNA. The larger products in the divergent cDNA reactions may represent non-specific or rolling-circle reverse transcription products [45]. On the other hand, PCR using convergent primers amplified products of the expected size when the template was cDNA from either time course or gDNA. We confirmed that the smaller product size in the case of convergent cDNA reactions corresponded to intron removal. (D) Sanger sequencing of divergent amplicons of the expected size confirmed the ARP_circRNA junction in both time courses. The extra GTAG in the predicted sequence marks the non-canonical ARP_circRNA splice junction (highlighted in red in the consensus sequence)