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An Accurate and Precise ddPCR-Based Method for Determining the Concentration of Plasmid DNA
Franco Puleo1, Annicka Evans1, Cullen Mason1
1Technical Development, Biogen, Cambridge, MA, USA.
Bio-Protocol
|July 28, 2026
Summary
A new digital droplet PCR (ddPCR) method accurately quantifies plasmid DNA for gene therapy production. This method overcomes limitations of traditional A260/A280 readings, ensuring reliable viral particle manufacturing.
Area of Science:
- Molecular Biology
- Gene Therapy
- Biotechnology
Background:
- Transient transfection of HEK293 cells with Rep/Cap, Helper, and gene-of-interest plasmids is crucial for recombinant adeno-associated viral (AAV) particle production in gene therapy.
- Accurate plasmid concentration assessment is critical for robust AAV production, but traditional A260/A280 spectrophotometric readings lack precision and specificity, being prone to matrix interference.
Purpose of the Study:
- To develop and validate a highly accurate and specific method for quantifying plasmid DNA concentration in the context of AAV production.
- To overcome the limitations of spectrophotometric plasmid quantification, particularly in complex sample matrices encountered during viral vector manufacturing.
Main Methods:
- A digital droplet PCR (ddPCR) workflow was established, incorporating a combined restriction digest and PCR protocol.
- This method linearizes plasmid DNA and quantifies specific gene copy numbers, enabling precise titer determination.
- The ddPCR assay was designed for universal applicability to plasmids with common selection cassettes, minimizing the need for redesign.
Main Results:
- The developed ddPCR method demonstrated high accuracy, specificity for plasmid DNA, and robustness against matrix interference.
- The assay exhibited high dilution tolerance, ensuring reliable quantification from crude cell lysates to purified plasmid preparations.
- Quantification was independent of contaminating nucleic acids (DNA, RNA) or dNTPs, providing accurate readouts where A260 measurements fail.
Conclusions:
- Digital droplet PCR offers a superior alternative to A260/A280 measurements for plasmid quantification in gene therapy manufacturing.
- This ddPCR method ensures precise and reliable plasmid titer assessment, critical for optimizing recombinant AAV production.
- The assay's specificity and robustness in complex matrices significantly improve the quality control of viral vector production.
