Fig. 3

Replication of DNA by its encoded proteins. a IVTTR reaction scheme using the oriLR-p2-p3 DNA template. Short amplification products are not represented. b The replication products of either the oriLR-p2-p3 or the p2-p3 DNA template (100 ng input) expressed in PUREfrex were visualized on agarose gel after RNase and Proteinase K treatments, followed by RNeasy clean-up column purification. The results from five independent replication experiments are shown in Supplementary Fig. 9a, Supplementary Fig. 10 and Supplementary Fig. 12b,e. In each IVTTR reaction triggered by the expression of the oriLR-p2-p3 DNA construct, 2.5 nM of template produced about 100 nM of p2 and 700 nM of p3 proteins (as estimated in Supplementary Fig. 3), which were able to generate ~50 nM of full-length DNA product when the reaction was supplemented with purified p5 and p6. c Samples were further incubated with λ-exonuclease to remove TP-uncapped DNA. The asterisk indicates full-length TP-capped DNA that has not been degraded by the λ-exonuclease. d De novo synthesized DNA was subsequently used as a template for a second IVTT reaction. The translation products were visualized by PAGE with GreenLys labeling. Expression of DNA that resulted from an IVTTR in the presence of purified p5 and p6 proteins led to fluorescent p2 and p3 protein bands of similar intensity as that measured when starting with 2.5 nM purified DNA (control with PCR product) demonstrating that the encoded functions are retained during amplification. Protein gels from two independent replication experiments are shown in Supplementary Fig. 9b and Supplementary Fig. 11. Note that the modest replication efficiency in the absence of purified p5 and p6 was sufficient to generate the encoded p2 and p3 proteins through amplification of information at the transcription and, to a lower extent, at the translation levels