This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Quantitative real-time polymerase chain reaction qPCR is a powerful technique that allows accurate and sensitive quantification of starting amounts of DNA without post-PCR manipulation [1].
It is also an essential technique for quantifying gene or noncoding DNA copy number in a cell [5] , [6]. Among the various types of standard DNA, plasmid DNA, especially the uncut circular one, is the most common choice due to its high stability and reproducibility.
It has been shown that uncut circular plasmid DNA is mostly in supercoiled form [8] , and that the supercoiled structure of the untreated template plasmid DNA can suppress real-time PCR compared to other relaxed templates [9].
However, the magnitude of error a circular plasmid standard may cause and what other conformational types of DNA can be a better choice of standard remain obscure. In this study, we evaluated three most common forms of standard DNA: circular plasmid, linearized plasmid digested by restriction enzyme , and linear PCR amplicon. Proliferating cell nuclear antigen gene pcna , a ubiquitous gene in eukaryotes, from four dinoflagellates and a diatom was used as the model gene for the study.
Quantification accuracies of real-time PCR assays based on different standards were compared. Consistently, significant differences were observed in the threshold cycle number Ct between the circular plasmid and linear linearized plasmid or linear PCR amplicon DNA. We further used these different conformational types of DNA as standard in qPCR to quantify the pcna copy number in the fully sequenced T. Our results demonstrated that the linear DNA standards including linearized plasmids, but not the circular plasmid standard, were reliable for absolute qPCR.
The monoclonal cultures of four harmful bloom-forming dinoflagellates and one fully sequenced diatom were used in this study. The dinoflagellate Alexandrium fundyense CA28 was provided by D. Anderson at Woods Hole Oceanographic Institution.
Cell concentrations were measured in triplicate using Sedgwick-Rafter counting chambers. The A. Other species used in this study had weak theca and hence the homogenization step was omitted. Total RNA was isolated as reported [12]. The full-length cDNA of K. Proliferating cell nuclear antigen gene pcna was chosen as the model in this study because it is a common gene in all eukaryotes and it is a target of our research as a potential cell cycle marker for algal growth rate studies [14].
For T. For A. For K. Clones were randomly picked and plasmid DNA was isolated from 2 ml of bacterial culture using the Qiaprep Spin Miniprep kit to avoid the contamination by bacterial RNA that may occur with a non-column-based plasmid isolation method. Circular plasmid and linear standards were compared to examine the effect of DNA structural confirmation on PCR result and amplification efficiency. In the A. In order to minimize the experimental error and test the plasmid purity, a second circular plasmid standard AfuC2 was prepared by further purifying AfuC1 using the Zymo DNA Clean and Concentrator kit.
Two linearized plasmid standards for A. In parallel, a linear PCR amplicon standard for A. Similarly, the circular plasmid standards for P. The linearized plasmid standards for K. The linear PCR amplicon standards of P. For this reason, plasmids can copy themselves independently of the bacterial chromosome, so there can be many copies of a plasmid — even hundreds — within one bacterial cell. Plasmids contain just a few genes, but they make a big difference to their host bacterium. Other plasmids contain genes that help the host to digest unusual substances or to kill other types of bacteria.
However, by protecting its bacterial host from stress-related death, a plasmid maximises its chances of being kept around. Under stressful conditions, bacteria with the plasmid will live longer — and have more opportunity to pass on the plasmid to daughter cells or to other bacteria. Bacteria without the plasmid are less likely to survive and reproduce. Some plasmids take extreme measures to ensure that they are retained within bacteria. Information S2. Information S3. Rarefaction type evaluation of sequencing depth.
Information S4. Acknowledgments We thank Karin P. References 1. Lederberg J Cell genetics and hereditary symbiosis. Physiol Rev — View Article Google Scholar 2. Nature Reviews Microbiology 3: — View Article Google Scholar 3. In: Francino MP, editors. Horizontal Gene Transfer in Microorganisms.
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Biophys J ;—7. Hunziker P. Nanomedicine: the application of the nanosciences to the benefit of the patient. European Journal of Nanomedicine ; Your documents are now available to view. Confirm Cancel. Accessible Published by De Gruyter December 2, From the journal European Journal of Nanomedicine.
Cite this. Abstract The ability to efficiently transfect plasmid DNA pDNA into eukaryotic cells has exerted major impact on scientific research in recent years, and translation to clinical application is ongoing, but challenging. Keywords: cytotoxicity ; DNA transfection ; polyethylenimine.
Figure 3 Comparison of transfection efficiency and cytotoxicity of circular and linearized plasmid DNA. We thank V. Received: Accepted: Published Online: Published in Print: Lehner, R. Plasmid linearization changes shape and efficiency of transfection complexes. European Journal of Nanomedicine , 5 4 , European Journal of Nanomedicine, Vol.
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