Figure 1. Schematic representation of the transcription- and translation-coupled DNA replication system.

Circular DNA encoding phi29 DNA polymerase under control of the T7 promoter is incubated with the reconstituted translation system including T7 RNA polymerase. mRNA is transcribed from the DNA, and phi29 DNA polymerase is translated. The polymerase attaches to the circular DNA and initiates the polymerization of a long single-stranded RNA in a rolling-circle manner. The polymerase further synthesizes the complementary strand to produce double-stranded DNA, which is a long repeat of the circular DNA sequence. The next round of transcription and translation occurs from the double-stranded DNA.