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Improved determination of plasmid copy number using quantitative real-time PCR for monitoring fermentation processes.

ABSTRACT: BACKGROUND: Recombinant protein production in Escherichia coli cells is a complex process, where among other parameters, plasmid copy number, structural and segregational stability of plasmid have an important impact on the success of productivity. It was recognised that a method for accurate and rapid quantification of plasmid copy number is necessary for optimization and better understanding of this process. Lately, qPCR is becoming the method of choice for this purpose. In the presented work, an improved qPCR method adopted for PCN determination in various fermentation processes was developed. RESULTS: To avoid experimental errors arising from irreproducible DNA isolation, whole cells, treated by heating at 95 degrees C for 10 minutes prior to storage at -20 degrees C, were used as a template source. Relative quantification, taking into account different amplification efficiencies of amplicons for chromosome and plasmid, was used in the PCN calculation. The best reproducibility was achieved when the efficiency estimated for specific amplicon, obtained within one run, was averaged. It was demonstrated that the quantification range of 2 log units (100 to 10000 bacteria per well) enable quantification in each time point during fermentation. The method was applied to study PCN variation in fermentation at 25 degrees C and the correlation between PCN and protein accumulation was established. CONCLUSION: Using whole cells as a template source and relative quantification considering different PCR amplification efficiencies are significant improvements of the qPCR method for PCN determination. Due to the approaches used, the method is suitable for PCN determination in fermentation processes using various media and conditions.

SUBMITTER: Skulj M

PROVIDER: S-EPMC2311272 | biostudies-literature | 2008

REPOSITORIES: biostudies-literature

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Improved determination of plasmid copy number using quantitative real-time PCR for monitoring fermentation processes.

Skulj Mihaela M Okrslar Veronika V Jalen Spela S Jevsevar Simona S Slanc Petra P Strukelj Borut B Menart Viktor V

Microbial cell factories 20080307

<h4>Background</h4>Recombinant protein production in Escherichia coli cells is a complex process, where among other parameters, plasmid copy number, structural and segregational stability of plasmid have an important impact on the success of productivity. It was recognised that a method for accurate and rapid quantification of plasmid copy number is necessary for optimization and better understanding of this process. Lately, qPCR is becoming the method of choice for this purpose. In the presente ...[more]

PMID: 18328094

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Project description:BackgroundCYP2D6 testing is increasingly used to guide drug therapy and thus, reliable methods are needed to test this complex and polymorphic gene locus. A particular challenge arises from the detection and interpretation of structural variants (SVs) including gene deletions, duplications, and hybrids with the CYP2D7 pseudogene. This study validated the Absolute Q™ platform for digital PCR-based CYP2D6 copy number variation (CNV) determination by comparing results to those obtained with a previously established method using the QX200 platform. In addition, protocols for streamlining CYP2D6 CNV testing were established and validated including the "One-pot" single-step restriction enzyme digestion and a multiplex assay simultaneously targeting the CYP2D6 5'UTR, intron 6, and exon 9 regions.MethodsGenomic DNA (gDNA) samples from Coriell (n = 13) and from blood, saliva, and liver tissue (n = 17) representing 0-6 copies were tested on the Absolute Q and QX200 platforms. Custom TaqMan™ copy number (CN) assays targeting CYP2D6 the 5'UTR, intron 6, and exon 9 regions and a reference gene assay (TERT or RNaseP) were combined for multiplexing by optical channel. In addition, two digestion methods (One-pot digestion and traditional) were assessed. Inconclusive CN values on the Absolute Q were resolved using an alternate reference gene and/or diluting gDNA.ResultsOverall, results between the two platforms and digestions methods were consistent. The "One-pot" digestion method and optically multiplexing up to three CYP2D6 regions yielded consistent result across DNA sample types and diverse SVs, reliably detecting up to 6 gene copies. Rare variation in reference genes were found to interfere with results and interpretation, which were resolved by using a different reference.ConclusionThe Absolute Q produced accurate and reliable CYP2D6 copy number results allowing for a streamlined and economical protocol using One-pot digestion and multiplexing three target regions. Protocols are currently being expanded to other pharmacogenes presenting with SVs/CNVs.

Project description:Bacteria can be considered as biological nanofactories that manufacture a cornucopia of bioproducts most notably recombinant proteins. As such, they must perfectly match with appropriate plasmid vectors to ensure successful overexpression of target genes. Among many parameters that correlate positively with protein productivity plasmid copy number plays pivotal role. Therefore, development of new and more accurate methods to assess this critical parameter will result in optimization of expression of plasmid-encoded genes. In this study, we present a simple and highly accurate method for quantifying plasmid copy number utilizing an EvaGreen single colour, droplet digital PCR. We demonstrate the effectiveness of this method by examining the copy number of the pBR322 vector within Escherichia coli DH5α cells. The obtained results were successfully validated by real-time PCR. However, we observed a strong dependency of the plasmid copy number on the method chosen for isolation of the total DNA. We found that application of silica-membrane-based columns for DNA purification or DNA isolation with use of bead-beating, a mechanical cell disruption lead to determination of an average of 20.5 or 7.3 plasmid copies per chromosome, respectively. We found that recovery of the chromosomal DNA from purification columns was less efficient than plasmid DNA (46.5 ± 1.9% and 87.4 ± 5.5%, respectively) which may lead to observed differences in plasmid copy number. Besides, the plasmid copy number variations dependent on DNA template isolation method, we found that droplet digital PCR is a very convenient method for measuring bacterial plasmid content. Careful determination of plasmid copy number is essential for better understanding and optimization of recombinant proteins production process. Droplet digital PCR is a very precise method that allows performing thousands of individual PCR reactions in a single tube. The ddPCR does not depend on running standard curves and is a straightforward and reliable method to quantify the plasmid copy number. Therefore we believe that the ddPCR designed in this study will be widely used for any plasmid copy number calculation in the future.

Project description:BackgroundAs compared with traditional transgene copy number detection technologies such as Southern blot analysis, real-time PCR provides a fast, inexpensive and high-throughput alternative. However, the real-time PCR based transgene copy number estimation tends to be ambiguous and subjective stemming from the lack of proper statistical analysis and data quality control to render a reliable estimation of copy number with a prediction value. Despite the recent progresses in statistical analysis of real-time PCR, few publications have integrated these advancements in real-time PCR based transgene copy number determination.ResultsThree experimental designs and four data quality control integrated statistical models are presented. For the first method, external calibration curves are established for the transgene based on serially-diluted templates. The Ct number from a control transgenic event and putative transgenic event are compared to derive the transgene copy number or zygosity estimation. Simple linear regression and two group T-test procedures were combined to model the data from this design. For the second experimental design, standard curves were generated for both an internal reference gene and the transgene, and the copy number of transgene was compared with that of internal reference gene. Multiple regression models and ANOVA models can be employed to analyze the data and perform quality control for this approach. In the third experimental design, transgene copy number is compared with reference gene without a standard curve, but rather, is based directly on fluorescence data. Two different multiple regression models were proposed to analyze the data based on two different approaches of amplification efficiency integration. Our results highlight the importance of proper statistical treatment and quality control integration in real-time PCR-based transgene copy number determination.ConclusionThese statistical methods allow the real-time PCR-based transgene copy number estimation to be more reliable and precise with a proper statistical estimation. Proper confidence intervals are necessary for unambiguous prediction of trangene copy number. The four different statistical methods are compared for their advantages and disadvantages. Moreover, the statistical methods can also be applied for other real-time PCR-based quantification assays including transfection efficiency analysis and pathogen quantification.

Dataset Information

Improved determination of plasmid copy number using quantitative real-time PCR for monitoring fermentation processes.

Publications

Improved determination of plasmid copy number using quantitative real-time PCR for monitoring fermentation processes.

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