Time to detect and fluorescence are proportional to DNA size and concentration. The limit of quantification for Nanodrop measurements was defined as 6 times the standard deviation of 10 true negative replicates [ 15 ]. The standard deviation of true negatives principle used to determine the LOQ for the Nanodrop is not applicable to Qubit measurements as DNA concentrations out of range of the standard curve are stated as such by the instrument and therefore no values are provided for true negatives [ 15 ].
Standard curves of plasmids containing PCR amplified target sequences were used for absolute quantification. Between 1 ng and 10 ng of extracted DNA was added to sample reactions, molecular grade water was added to the no template control reactions in place of extracted DNA, and a 10 fold plasmid dilution series was added to standard curve reactions. Primer concentrations were optimized according to Nolan et al.
Reaction specificity was verified using melt curve analysis, and r 2 values were determined using SDS v2. Except for two of the B. Extraction efficiency was calculated Eq. Extraction methods could not be compared due to unknown day-to-day variability in DNA extraction efficiency. ANOVA analysis was run on datasets without outliers. The dataset supporting the results of this article is included as an additional file to the manuscript Additional file 5. NDO and JMB conceived of the study and its design, performed the data analysis, and wrote the manuscript.
NDO conducted the experiments. Both authors read and approved the final manuscript. Table S1. Figure S1. The experiments were executed on two separate days, A and B: with single 4 runs each and triple 4 runs each replicates respectively.
Figure S2. The two manufacturer supplied markers are at 50 bp and bp. Dotted lines indicate the bp point on the x-axis. The bottom number within each graph is the number of replicates where the area under the curve for the full electropherogram was above the analysis threshold. The top two numbers are the mean and standard deviation for the percentage of DNA that was greater than bp top value and less than bp middle value. Scales are independent for each row due to the large range in responses.
Figure S3. Three dilution series replicates were processed, indicated in red, green, and blue, with five runs each. Dotted line indicates the defined limit of quantification 0. PDF 94 kb. Raw data sheet labeled "DNAcompleteResults. The authors would like to acknowledge Dr. Autumn Downey, Dr. Sandra da Silva, Jennifer McDaniel, and Lindsay Vang for assistance and guidance on the planning and execution of the project. Additionally, we would like to thank Drs.
Erica Seifert and Sandra Da Silva for their comments and assistance during the writing process; as well Dr. David Duewer and Margaret Kline and the anonymous reviewers for reviewing the manuscript. Thanks to Dr. National Center for Biotechnology Information , U. BMC Res Notes. Published online Dec 3. Author information Article notes Copyright and License information Disclaimer. Corresponding author. Nathan D Olson: vog. Received Jul 25; Accepted Nov This article has been cited by other articles in PMC.
Additional file 2 Figure S1. Additional file 3 Figure S2. Additional file 4 Figure S3. Abstract Background Quantitative polymerase chain reaction qPCR assays used in pathogen detection require rigorous methods development including characterizing DNA extraction products. Results DNA extract quantity concentration and extraction efficiency and quality purity and intactness varied by cell type and extraction method enabling the demonstration of different DNA characterization methods.
Conclusion The presented DNA extract characterization process provides measures of DNA quantity and quality applicable to microbial detection methods development and validation studies. Background Developments in molecular based microbial detection methods, such as quantitative polymerase chain reaction qPCR [ 1 ], have significantly contributed to rapid identification and quantification of unknown biological agents [ 2 ]. Results DNA extracted from five different cell types using six different extraction methods Table 1 was applied to DNA characterization methods as shown in Figure 1.
Open in a separate window. Figure 1. Table 1 Extraction methods by application and processes. Figure 2. Figure 3. Figure 4. Discussion The purpose of this work was to evaluate commonly used DNA characterization methods for applicability to inform the assay developer of DNA extraction method performance.
DNA quality DNA extract purity is of interests in terms of how contaminants will affect downstream assay performance. DNA quantity Accurate DNA quantity measures are critical to assay optimization as losses in DNA due to extraction procedures contribute to a reduction in overall detection assay performance [ 25 , 38 ]. Conclusions During optimization of detection assays and determining which DNA extraction method to use, downstream application requirements and common sources of downstream application inhibitors [ 55 - 57 ] will dictate what methods are chosen to characterize DNA extract quality and quantity.
Availability of supporting data The dataset supporting the results of this article is included as an additional file to the manuscript Additional file 5. Supplementary Material Additional file 1: Table S1. Click here for file 43K, doc. Additional file 2: Figure S1. Click here for file K, pdf. Additional file 3: Figure S2. Click here for file 1. Additional file 4: Figure S3. PDF 94 kb Click here for file 94K, pdf.
Click here for file 30K, xlsx. Acknowledgements The authors would like to acknowledge Dr. Real-time PCR in clinical microbiology: applications for routine laboratory testing. Clin Microbiol Rev. The challenge to quantify Listeria monocytogenes --a model leading to new aspects in molecular biological food pathogen detection. J Appl Microbiol. Molecular diagnostics: harmonization through reference materials, documentary standards and proficiency testing.
A further option in the new MARS data analysis software is the possibility to determine the DNA concentration of unknown samples without a standard curve. It should be taken into account that this method only works well when the path length correction feature is activated. Furthermore, with the help of the data analysis software, MARS, it is possible to determine different nucleic acid concentrations depending on the extinction coefficient.
A full absorbance spectrum in the range of nm helps to identify impurities and it can be measured within one second per well. Download application note pdf. High degree of linearity from 0. Introduction One of the most common methods for nucleic acid detection is the measurement of solution absorbance at nm A due to the fact that nucleic acids have an absorption maximum at this UV wavelength.
Also interesting for you Read more about absorbance-based detection and our absorbance microplate readers. The ratio between the absorbances at A and nm A is broadly accepted as a means of assessing protein contamination in a sample of purified DNA. In this example, our DNA concentration would actually be only half the concentration calculated by A While some systems offer a corrected DNA concentration based on the deviation of the sample's spectrum from the theoretical spectrum of pure DNA, the high amounts of protein required for the difference to be measurable make this correction unreliable.
Proteins are not the only possible contaminant in purified DNA samples. This means that it is not possible to assess the purity of DNA samples below this concentration by using this method. This is due to the fact that measured values are very close to the detection limit of the instrument, where the variability of the measurement compared to the measured values is enormous, and the A and A values are even lower than those of A , and hence closer to the detection limit of the instrument.
But an oscillation of just 0. Therefore, it is not uncommon for ratios to give even negative values because of A or A going below 0. Enter your username and we'll send a link to reset your password. Username Username not found. Send Email. A password reset email has been sent to the primary email address associated with your account.
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