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Fig. 2 | Virology Journal

Fig. 2

From: An optimized circular polymerase extension reaction-based method for functional analysis of SARS-CoV-2

Fig. 2

Cloning-free generation and characterization of CPER-derived recombinant SARS-CoV-2. A Representative gel images of the overlapping cDNA fragments amplified from purified SARS-CoV-2 genomic RNAs of the indicated virus strains. The primer sets described by Torii et al. [26] conform to the genome sequences of the ancestral strain (WA1) and the selected Beta and Omicron variants of concern (VOCs) with 100% complementarity. MM, molecular marker; kb, kilobase. B Schematic of the overlapping PCR strategy for site-directed mutagenesis in fragment 2 by using purified PCR product as a template (top panel), as well as representative gel images of the intermediate (2.1 and 2.2) and final (2mut) PCR products (bottom panel). MM, molecular marker; Omic, Omicron. C Sequencing confirmation of the integrity of the spike furin cleavage site of the passage 0 (P0) virus stocks from three independent virus rescues (#1 – #3) using the optimized CPER approach. aa, amino acids; nt, nucleotides. D Plaque morphology on Vero E6-TMPRSS2 cells of recombinant Beta (rBeta) generated by optimized CPER as well as of its parental virus. E Virus titers of the P0 stocks of CPER-derived recombinant WA1 (rWA1) and rBeta, collected at day 5 and day 4 post-transfection of the CPER product, respectively (n = 4 independent CPER rescues)

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