Figure 2. Production of linear, covalently-closed minimalistic expression DNA constructs.

(A) Production of MIDGE DNA. The purified parental plasmid containing the GOI flanking with a restriction site (e.g. EcoRI) is digested by the RE. The resulting fragments are ligated to the hairpin oligodeoxynucleotides to generate MIDGE DNA molecules. Then, unligated fragments including plasmid backbones are digested by T7 DNA polymerase and the MIDGE DNA is purified. (B) Production of Doggybone™ DNA. The purified parental plasmid containing the GOI flanking with telomeric ends (Tel-L and Tel-R) is denatured by NaOH and used as a template DNA in a RCA reaction using Phi29 DNA polymerase. The resulting DNA concatemers are cleaved and joined by an enzymatic reaction using TelN protelomerase to generate Doggybone DNA. Doggybone™ DNA molecules are then purified using chromatographic purification. The purified Doggybone DNA may be used as a template DNA for the RCA reaction.

GMO: Genetically modified organism; GOI: Gene of interest; MIDGE: Minimalistic, immunologically defined gene expression; RCA: Rolling circle amplification.