Recombination efficiency measurement by real-time PCR: A strategy to evaluate ParA-mediated minicircle production
Cláudia P A Alves1, Duarte Miguel F Prazeres1, Gabriel A Monteiro1
1iBB - Institute for Bioengineering and Biosciences, Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001, Lisboa, Portugal; Associate Laboratory i4HB - Institute for Health and Bioeconomy at Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal.
Abstract:
Minicircles (MCs) are DNA molecules that are produced in Escherichia coli by replicating a parental plasmid (PP) and inducing its site-specific intramolecular recombination into miniplasmid (MP; containing the prokaryotic backbone) and MC molecules (comprised by the eukaryotic cassette). The determination of the recombination efficiency and the monitoring of PP, MC and MP species during processing and in the final product are critical aspects of MC manufacturing. This work describes a real-time PCR method for the specific identification of PP, MP or MC that uses sets of primers specific for each species. The method was evaluated using artificial mixtures of (i) PP and MP, (ii) PP and MC and (iii) MP and MC that were probed for all three DNA molecules. The ratio of molecules of each DNA species in these mixtures were determined with differences lower than 10% relatively to the expected ratio of the species in 90% of the mixtures. Next, the recombination efficiency was successfully estimated by analysing pre-purified DNA samples obtained from cell cultures. A standard deviation < 2% was obtained between replicas and results closely correlated with those obtained by densitometry analysis of agarose gels. Further optimization is required to determine recombination efficiency directly from whole cells.
Insights
A new real-time PCR method accurately quantifies DNA molecules (parental plasmid, miniplasmid, minicircle) crucial for manufacturing. This technique aids in determining recombination efficiency for improved minicircle DNA production.
Area of Science:
- Molecular Biology
- Biotechnology
- DNA Manufacturing
Background:
- Minicircles (MCs) are vital DNA molecules produced via recombination of parental plasmids (PP) in Escherichia coli.
- Monitoring parental plasmid (PP), miniplasmid (MP), and minicircle (MC) DNA species is critical for efficient MC manufacturing.
- Accurate determination of recombination efficiency is essential for optimizing MC production processes.
Purpose of the Study:
- To develop and evaluate a real-time PCR method for specific identification and quantification of PP, MP, and MC DNA.
- To assess the method's accuracy in determining the ratios of different DNA species in artificial mixtures.
- To estimate recombination efficiency using the developed real-time PCR assay on purified DNA samples.
Main Methods:
- Design of species-specific primer sets for real-time PCR targeting PP, MP, and MC DNA.
- Validation using artificial mixtures of PP/MP, PP/MC, and MP/MC DNA.
- Analysis of pre-purified DNA samples from cell cultures to determine recombination efficiency.
Main Results:
- The real-time PCR method accurately identified and quantified PP, MP, and MC DNA species.
- Quantification of DNA species ratios in artificial mixtures showed less than 10% difference from expected values in 90% of cases.
- Recombination efficiency estimation yielded high reproducibility (standard deviation < 2%) and correlated well with gel densitometry.
Conclusions:
- A novel real-time PCR assay provides a reliable tool for monitoring DNA species during minicircle production.
- The method enables accurate determination of recombination efficiency from purified DNA, crucial for process control.
- Further optimization is needed to adapt the assay for direct analysis from whole cells.
