FIG. 3.

Inverse nested PCR of right-hand virus-cell junctions. Determination of the limiting dilution for inverse nested PCR of cellular DNA (A) and the products from 22 individual limiting dilution amplifications (B) are shown. (A) Cellular DNA from wild-type-virus-infected duck 10 was processed as described in the legend to Fig. 2A and added to individual amplification reaction mixtures in the amounts indicated. After two rounds of PCR with nested primers, the products were analyzed by electrophoresis through a 1.5% agarose gel and stained with ethidium bromide. The absence of products in the reactions containing less than 500 pg of cellular DNA indicates that amplifiable junction fragments were not present in these dilutions. The presence of heterogeneous products in reactions containing more than 500 pg of cellular DNA indicates that multiple junction fragments were present. The two discrete products in the 500-pg reaction indicate that two junction fragments were present. (B) Twenty-two individual reactions (indicated above the lanes), each containing 100 pg of cellular DNA from the reaction shown in panel A, were assayed for the presence of discrete products. Nine unique products were produced in eight of the reactions. Therefore, the frequency of junction fragments was nine per 2.2 ng of cellular DNA. The PCR products were characterized by direct sequencing. The product from reaction 20 was unsuitable for sequencing because of the mixed product. Lane m, molecular weight markers of bacteriophage λ DNA digested with HindIII.