Real-Time PCR Determination of Escherichia coli Genomic DNA Contamination in Plasmid Preparations

Vilalta, Adrian; Whitlow, Vanessa; Martin, Terrie

doi:10.1006/abio.2001.5480

Cited by 13 publications

(3 citation statements)

References 8 publications

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“…The accumulation of primer dimers and the amplification of non-specific PCR products can be analyzed only in SYBR Green I detection (Deprez et al, 2002). In a previous study, SYBR Green I detection was used to determine host DNA contamination in plasmid preparations and provided a reliable alternative to traditional blotting methods and to more expensive TaqMan protocols (Vilalta et al, 2002).…”

Section: Discussionmentioning

confidence: 99%

Absolute and relative QPCR quantification of plasmid copy number in Escherichia coli

Lee

Kim

Shin

et al. 2006

Journal of Biotechnology

570

401

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Real-time QPCR based methods for determination of plasmid copy number in recombinant Escherichia coli cultures are presented. Two compatible methods based on absolute and relative analyses were tested with recombinant E. coli DH5␣ harboring pBR322, which is a common bacterial cloning vector. The separate detection of the plasmid and the host chromosomal DNA was achieved using two separate primer sets, specific for the plasmid ␤-lactamase gene (bla) and for the chromosomal d-1-deoxyxylulose 5-phosphate synthase gene (dxs), respectively. Since both bla and dxs are single-copy genes of pBR322 and E. coli chromosomal DNA, respectively, the plasmid copy number can be determined as the copy ratio of bla to dxs.These methods were successfully applied to determine the plasmid copy number of pBR322 of E. coli host cells. The results of the absolute and relative analyses were identical and highly reproducible with coefficient of variation (CV) values of 2.8-3.9% and 4.7-5.4%, respectively. The results corresponded to the previously reported values of pBR322 copy number within E. coli host cells, 15-20.The methods introduced in this study are convenient to perform and cost-effective compared to the traditionally used Southern blot method. The primer sets designed in this study can be used to determine plasmid copy number of any recombinant E. coli with a plasmid vector having bla gene.

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Section: Discussionmentioning

confidence: 99%

Absolute and relative QPCR quantification of plasmid copy number in Escherichia coli

Lee

Kim

Shin

et al. 2006

Journal of Biotechnology

570

401

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“…P7589) 10 . Further, real-time quantitative PCR (RT-PCR) was employed to detect the presence of any residual E. coli genomic DNA in the plasmid preparation 11 . Brie y, we designed forward (5'-AAGCTGCCTGCACTAATGTTCC-3') and reverse (5'-TCGCGTACCGTCTTCATGG-3') primers for the ampli cation an amplicon of the E. coli genome.…”

Section: Plasmid Preparationmentioning

confidence: 99%

Robust and rapid pipeline for the development of mRNA vaccine for the fast-emerging SARS-CoV-2 variants

Soni¹,

Khanzode²,

Srivast³

et al. 2022

Preprint

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Recently, genomics has gained a lot of attention in the field of bio-therapeutics, vaccines, and gene therapy. We have developed a robust pipeline for establishing the mRNA vaccine portfolio to address the current pandemic and endemic of COVID-19, infectious diseases, and cancer. We have designed and assembled approximately 4000 base pair DNA encoding the spike protein of the SARS-CoV-2 Omicron variant (B.1.1.529) using overlapping oligo-based PCR assembly. Further, we cloned this fragment into a self-amplifying mRNA platform and prepared the messenger RNA (mRNA). The integrity and sequence were confirmed through multiple orthogonal techniques such as capillary electrophoresis, Sanger DNA sequencing, and Next-generation sequencing for mRNA. The mRNA was transfected into HEK 293T cells and a significant expression of the spike protein was monitored by FACS. Herein, we propose a robust and rapid pipeline for the development of mRNA for vaccines starting from sequence analysis to identifying the lead candidate within 4–5 weeks.

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“…Chromosomal DNA contamination in plasmid-purified samples was measured via QPCR. Oligonucleotide primers (Qiagen) 23S-sense (5 -GAAAGGCGCGCGATACAG-3 ) and 23S-antisense (5 -GTCCCGCCCTACTCATCGA-3 ) were used to amplify a 70-bp fragment of the 23 S rRNA gene present in the E. coli genome [17]. Purified E. coli DH5α genomic DNA was denatured at 95 • C for 15 min and cooled on ice prior to use in amplification reactions to generate standard curves from 1 pg to 5 ng.…”

Section: Qpcr (Quantitative Pcr) Assaymentioning

confidence: 99%

Large‐scale plasmid DNA processing: evidence that cell harvesting and storage methods affect yield of supercoiled plasmid DNA

Kong¹,

Rock²,

Booth³

et al. 2008

Biotech and App Biochem

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The effect of bacterial-cell centrifugation and handling on the initial stages of plasmid processing was investigated. Escherichia coli cells containing either a 6 or 20 kb plasmid were grown in 75- and 450-litre bioreactors, and the process yield of the early recovery stages was characterized in terms of SC pDNA (supercoiled plasmid DNA) recovered. In all cases, the cells were totally recovered using either a continuous-feed, intermittent-solids-discharge, disc-stack centrifuge or a continuous-feed, batch-discharge, solid-bowl centrifuge. The cells were then either processed immediately or stored frozen. The centrifugation method considerably affected the yield of SC pDNA, and there was evidence that the intermittent discharge of cells from a centrifuge operating at high speed led to a sediment containing lysed cells and degraded pDNA. This led to estimated plasmid yield losses of up to 40% as compared with cells recovered from laboratory or solid-bowl centrifuges, where there is evidently no cell stress on discharge. By inference, the cell stress on feed to either of the continuous centrifuges studied was not implicated in product loss. Freezing of the recovered cells gives a convenient hold stage prior to further processing. In all cases, this extra freeze-thaw stage led to loss of SC pDNA, and this was in addition to the loss attributed to cell lysis during centrifugation discharge. Only average yields can be gained from pilot plant-scale studies; separate laboratory-based experiments indicated that this loss of SC pDNA is determined by the time and temperature for which the resuspended cells are held.

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Real-Time PCR Determination of Escherichia coli Genomic DNA Contamination in Plasmid Preparations

Cited by 13 publications

References 8 publications

Absolute and relative QPCR quantification of plasmid copy number in Escherichia coli

Absolute and relative QPCR quantification of plasmid copy number in Escherichia coli

Robust and rapid pipeline for the development of mRNA vaccine for the fast-emerging SARS-CoV-2 variants

Large‐scale plasmid DNA processing: evidence that cell harvesting and storage methods affect yield of supercoiled plasmid DNA

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