FIG. 1.

Clonal frequency analysis by SSCP. A 185-bp fragment in the NS5A gene of HCV was amplified by PCR, and the PCR product was cloned into the pTAg vector. Thirty clones were isolated, clonal DNA was amplified by PCR, and the PCR products were analyzed by SSCP. Each clone theoretically gives two bands in SSCP, corresponding to the two strands of double-stranded PCR products, which migrate to positions depending strictly on their nucleotide sequence. At the low temperature used for migration (3°C), minor conformations of DNA strands can generate weak additional bands which also migrate to specific positions. A first SSCP round was performed in random order. The second SSCP round, in which the clones are in the order of frequency deduced from the first analysis, is shown. Each lane corresponds to a different NS5A clone. After clonal frequency analysis, one to three (when available) clones per SSCP pattern were sequenced.