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. 2020 Jul;26(7):888–901. doi: 10.1261/rna.074963.120

FIGURE 2.

FIGURE 2.

Schematic diagram of DeSCo-PCR. (A) First-strand cDNA synthesis (red line) using template-specific reverse primer (RP) annealed to viral positive-strand RNA (bold black line). (B) Primer schematics indicating annealing of BP mostly upstream but extending downstream from the 5′ end of sgRNA sequence and annealing of FP to a longer tract starting exactly at the 5′ end of sgRNA sequence. This allows BP to win the annealing competition for gRNA and FP to win the annealing competition for the 5′ end of sgRNA. The dashed box shows the sequences of BP and FP primers and the partial cDNA sequences of RCNMV RNA1 and SR1f to which the primers anneal. (C) Primer competition at annealing step and subsequent extension step of DeSCo-PCR. Vertical lines represent base-pairing between the primers and the cDNA template. Circled X indicates primer that does not anneal in the presence of competing primer. (gRNA) genomic RNA, (sgRNA) subgenomic RNA, (FP) forward primer, (BP) blocking primer, (CR) competitive region (blue letters), (BR) blocking region (red letters), (NER) nonextendable region (green letters).