Use plasmid as pcr template

The enormous utility of PCR is based on its ease of use and its ability to amplify DNA in relatively short time periods. The PCR process uses an enzyme known as Taq polymerase, or variants of this enzyme that are called for in specific circumstances.

Taq polymerase is purified from a bacterium originally isolated from hot springs and is stable at the very high temperatures used for denaturation separation of DNA strands, typically C. In reality, the efficiency of amplification of a given enzyme preparation is somewhat less than ideal. PCR is performed in a thermal cycler, which is programmed to rapidly heat, cool and maintain samples at designated temperatures for varying amounts of time, determined by the investigator.

Master Mixes: Prepare master mix on ice , and keep it there! Think about why we should include these controls. For genes A and F, you would need Master Mix for four samples minimum including the provided positive control DNA , so you should prepare enough MM for FIVE samples always figuring that there will be some loss of reagent to the sides of the tube and the inside of pipette tips. It has two independently controlled well heating blocks.

Despite their good safety profile, evidence indicates that AAV vectors can trigger an inflammatory response in different tissues, including immune privileged organs, such as the eye [ 23 , 24 , 25 , 26 , 27 ]. One of the main factors that can contribute to toxicity is the vector dose. The determination of the minimal effective and non-toxic dose of a candidate vector in animal models before clinical trials is of vital importance in ensuring the success of gene therapy.

Without an accurate assay, it is not possible to correctly control the dose that subjects are given and can make the data generated from potency assays variable and difficult to interpret. To that effect, the accurate and reliable titration of the recombinant AAV particles is an essential prerequisite.

However, the accuracy of this method will depend on the experimental design and performance. The first step is to extract the viral DNA from the AAV particles through the enzymatic digestion of the capsid or the use of high temperatures. This was a surprising finding and contradictory to other studies that have shown an increase or an unchanged vector titer when using proteinase K treatment [ 16 , 31 ].

Nevertheless, the observed titer difference between supercoiled and linearized plasmid standards was maintained. It is possible that the proteinase K was not sufficiently inactivated in this study, as SDS is commonly applied to achieve this, whereas our protocol relied on heat inactivation. Identifying the specific cause of the difference would require extensive further investigations, but, for now, it is of interest to note that this appears to be the first presentation of such a finding.

The conformation of the DNA used to elaborate the standard curves is one of the variables that can impact the qPCR assay performance.

The standard curve included in a qPCR assay is calculated using two variables: the slope, which is a measure of the reaction efficiency, and the Y-intercept, which is the theoretical limit of detection of the reaction.

The supercoiled topological form that is characteristic of a plasmid DNA molecule is a twisted and winded circular DNA molecule that may exist in variable conformations, which would likely alter at different temperatures. This could influence the access of the PCR primers to the target sequence and, therefore, cause variation in the efficiency of primer binding and, consequently, target amplification.

This could result not only in increased variability, but also in a lower amount of DNA that is accessible for each round of amplification, which has an impact on the standard curve parameters with a similar slope, but usually higher Ct values. Hence, the quantification of the viral vector-derived DNA using a calibration curve that was prepared with a DNA whose conformation is supercoiled or twisted in a way that makes the target regions less accessible would lead to less reliable results.

Indeed, overestimation in qPCR has been reported that is caused by using supercoiled DNA as calibration standards [ 32 , 33 ]. The topology of this molecule prevents the correct performance of the qPCR, which, besides causing an overestimation in the absolute titers, contributes to increasing the variability in the results.

This variability not only leads to the inaccuracy of AAV titer calculations, but also to the impossibility to cross-compare individual AAV samples using a correction factor. In this study, we assessed the physical titer of an AAV.

This enzyme makes one cut in the plasmid DNA, with the consequent linearization of the plasmid. Significant differences are observed when the use of supercoiled plasmid or the HindIII -linearized plasmid as standard are used. In accordance with other studies, the viral genome titers are overestimated when using supercoiled plasmid as the standard.

The results obtained in this study suggest that the use of supercoiled plasmid as standard not only can affect the accuracy of the measure, but also the reproducibility of the results.

It is critical to reliably and accurately titer AAV preparations to ensure appropriate dosing is achieved in clinical trials and the treatment of patients. RPGR titres. One possible reason for this discrepancy is that linearized DNA may be easier for primers to bind to compared with supercoiled plasmids. All AAV publications should state which form of reference plasmid has been used to calculate titres, so that appropriate comparisons relating to dose toxicity and transduction efficacy can be made.

Conceptualization: C. Supervision: R. All authors have read and agreed to the published version of the manuscript. No competing financial interests exist for C. National Center for Biotechnology Information , U. Journal List Genes Basel v. Genes Basel. Published online Apr McClements , 1 and Robert E. MacLaren 1, 2. Michelle E. Find articles by Michelle E. Robert E. Author information Article notes Copyright and License information Disclaimer. Received Feb 22; Accepted Apr This article has been cited by other articles in PMC.

Abstract The ability to accurately determine the dose of an adeno-associated viral AAV therapeutic vector is critical to the gene therapy process. Keywords: adeno-associated virus, gene therapy, quantitative PCR. Introduction Adeno-associated virus AAV is a small non-enveloped virus that depends on other viruses, such as adenovirus, herpes virus, or papilloma virus to complete its life cycle and replication. Materials and Methods 2.

Open in a separate window. Figure 1. Table 1 Primers and probes designed to quantify AAV. RPGR titre. Statistical Analysis GraphPad Prism software version 7. Results 3. Figure 2. Figure 3. Discussion AAV-based vectors have been widely used in gene therapy in animal models and human clinical trials due to their high efficiency driving a long-lasting transgene expression and its low immunogenicity and pathogenicity [ 21 , 22 ].

Author Contributions Conceptualization: C. Institutional Review Board Statement Not applicable. Too high primer concentrations increase the chance of mispriming, which results in nonspecific PCR products.

Limiting primer concentrations result in extremely inefficient PCR reactions. The primer concentration can be calculated as described in Preparation of oligo solutions.

DNA template concentration. The concentration of DNA template depends on the source. Concentration of dNTPs.

DNA polymerase. Different polymerases are commercially available and are summarized in DNA polymerases. Polymerase buffer. All DNA polymerases are supplied with their own optimal polymerase buffer.