Supplementary Figure S4: Improved SARS-COV-2 PCR detection and genotyping with double-bubble primers

D-B primers proof of principle with GAPDH: end-point PCR utilizing RNA extracted from Huh cells. Lanes 2, 3- RNA crude prep; lanes 4, 5- RNA crude prep digested with DNase I; lanes 6, 7- RNA crude prep digested with DNase I and subjected to reverse-transcription. All preparations were amplified by PCR using normal (lanes 2, 4, 6; amplicon 135 bp) and D-B (lanes 3, 5, 7; amplicon 152bp) GAPDH primers and resolved on 1% agarose gel. Lane 1- 100 bp ladder. Note amplification using crude RNA prep, presumably due to amplification of GAPDH pseudogene in the residual genomic DNA and no amplification in RNA crude prep after DNase I digestion. Note also higher amplicon intensity of the D-B primers on the agarose gel due to bigger size amplicon. PCR was performed using standard conditions.