Home Resources Video Library One-day Workflow for synthesizing dbDNA™ using the EnClose Cell-free dbDNA™ Synthesis Kit

One-day Workflow for synthesizing dbDNA™ (doggybone™ DNA) using the EnClose™ Cell-free dbDNA Synthesis Kit

This animation demonstrates how to generate dbDNA (doggybone) using the EnClose Cell-free dbDNA Synthesis Kit.

Script

The EnClose™ Cell-free dbDNA™ Synthesis Kit includes everything needed to enzymatically generate closed-ended linear double-stranded DNA containing a sequence of interest. This kit can be used to create high-quality dbDNA for use in downstream applications such as in vitro transcription (IVT), lentiviral (LV) and adeno-associated virus (AAV) payloads, and more. First, a sequence of interest is cloned into the provided dbDNA Vector. After cloning, telRL sites flank the sequence of interest. These sites are crucial for TelN protelomerase activity and dbDNA formation. The sequence of interest residing in the vector now becomes the input plasmid for the dbDNA workflow. A small amount of input plasmid is amplified by phi29-XT DNA polymerase via rolling circle amplification, or RCA. When combined with the provided specific primer mix and dNTPs, the polymerase generates many copies of the input DNA within 3 hours of incubation. The provided forward and reverse primers allow the polymerase to generate linear double-stranded DNA concatemer repeats of the sequence of interest and the vector backbone. Next, TelN protelomerase covalently closes the ends flanking the sequence of interest as well as the remaining backbone of the input plasmid. TelN does this by cleaving its recognition sequence, thereby creating covalently-closed ends and forming two dbDNA products. One product contains the sequence of interest, and the other contains the remaining plasmid backbone. Adding a restriction enzyme that cuts only within the backbone sequence and not within the sequence of interest, such as XbaI, opens the backbone-containing dbDNA. This then allows T5 exonuclease to remove the unwanted sequence, while leaving the protected sequence of interest intact. After an overnight incubation, only the desired dbDNA product remains. Finally, a cleanup step using nucleic acid purification kits such as Monarch Nucleic Acid Purification Kits, removes unincorporated dNTPs, primers and enzymes, and leaves the dbDNA product ready for downstream applications.

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