The in vitro-amplified short DNAs replicate by a TP-priming mechanism. (A) Amplification assays were carried out as described, using either 50 ng (lanes 1 and 2) or 5 ng (lanes 3 and 4) of φ29 TP-DNA as input template, in the absence of SSB. After incubation for 90 min at 30°C, half of the reaction was processed as described in Fig. 2, and analyzed by native agarose gel electrophoresis followed by ethidium bromide staining. (B) The amplified DNA corresponding to lane 4 in A was 10-fold diluted and used as new input template for a second amplification assay. Reamplification was allowed for 90 min at 30°C, either with no addition (lane 1) or with the fresh addition of the indicated replication proteins (lanes 2 and 3). The reamplified DNA was treated with proteinase K and analyzed by native agarose gel electrophoresis followed by ethidium bromide staining.