Fig. 4.

Detection of the rare ZP6248.E321G mutation during acute infection. The presence of the ZP6248.E321G mutation (A to G) was examined by parallel allele-specific sequencing (PASS) analysis in plasma collected at the 9 March time point. In this assay, cDNA annealed to an acrydite-modified primer is immobilized in an acrylamide gel, after which in-gel PCR is performed, with the resulting products accumulating around the individual cDNA templates. Sequencing primers that anneal just upstream of the mutation site in V3 (GAA->GGA) can be used to distinguish wt and mutant bases at the same position by single-base extension using Cy3- and Cy5-labeled adenosine (wt) and guanosine (mutant), respectively. The gel was scanned to obtain images with a GenePix 4000B microarray scanner, and the spot number was counted using the Progenesis PG200 software. Green and red spots indicate wt and mutant bases, respectively, detected in individual viral genomes. Two mutants were identified by arrows. A partial image from one of the three experiments is shown.