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Self-circularized Overhang-based Unrecombined Products (SOUP): a rapid in vitro method for generating backbone-free
Ionuț Adrian Cepleanu-Pascu1,2, Mirel Adrian Popa3, Gheorghe Dănuț Cimponeriu1
1Faculty of Biology, University of Bucharest, Bucharest, Romania.
Biotechniques
|March 14, 2026
Summary
A new method called Self-circularized Overhang-based Unrecombined Products (SOUP) rapidly generates backbone-free DNA expression cassettes from PCR products. SOUP constructs show improved gene expression efficiency and transfection compared to traditional plasmids.
Area of Science:
- Molecular Biology
- Biotechnology
- Gene Expression
Background:
- Plasmid DNA is standard for mammalian transfection but has bacterial sequences impacting expression and safety.
- Minicircle DNA improves expression but requires complex, time-consuming protocols.
Purpose of the Study:
- To introduce Self-circularized Overhang-based Unrecombined Products (SOUP), a rapid workflow for creating backbone-free expression cassettes.
- To evaluate SOUP's efficiency and gene expression compared to traditional plasmids.
Main Methods:
- SOUP workflow involves PCR amplification with overhangs, type IIS restriction digestion, self-ligation, and RecBCD exonuclease treatment.
- Generated SOUP constructs were compared to a parental plasmid with an identical GFP cassette in HEK293 cells.
Main Results:
- SOUP produced circular expression cassettes in under 24 hours.
- At 56 hours post-transfection, SOUP showed a >2.5-fold higher productivity index, increased mean fluorescence, and higher transfection efficiency than plasmids.
- SOUP constructs demonstrated strong gene expression despite heterogeneous composition.
Conclusions:
- SOUP offers a fast, inexpensive, and practical alternative to plasmid vectors for generating backbone-free DNA constructs.
- The method serves as a valuable tool for rapid prototyping and enhancing gene expression in mammalian systems.
