Why is dna quantified at 260 nm

All extraction methods had low proportions of extracts within the accepted ratio. The reference method and precipB had purity ratios within or near the accepted ratios, 2. A high proportion E. For inhibited samples the expected difference in C t values between the control and inhibition reactions is greater than 1 cycle, therefore no inhibition was observed.

The mean C t values for the B. Evaluation of PCR inhibition for extracts from five cell types. Shape and color indicate the source cell type, B. Finally, DNA shearing was evaluated using microfluidic gel electrophoresis. Thirty-five samples had FC values above the cutoff with Slight shearing was observed for some of the vegetative cell samples extracted with methods using bead beating during the lysis step Additional file 3 : Figure S2. For example one B. For extracts from vegetative cells the reference method, which included a bead beating lysis step, produced fragments less than bp in size comprising 1.

The actual copy number LOQ for the three assays ranged from to copies per reaction. Overall DNA concentration ranged from Calculation of extraction efficiencies using qPCR concentration values allow for a more direct comparison with the literature where it is common to present extraction methods in terms of the overall process efficiency. Extraction efficiencies in general were higher for the two Gram-negative organisms than the hard to lyse cells types; yeast, spores, and Gram-positive.

The three precip extraction methods all used bead beating for lysis with precipitation and silica spin columns for purification but had different ranges in extraction efficiency Figure 4 C,D,F. Cell types are labeled as follows: B. Plots are grouped by extraction method. Scales are independent for each graph due to the large range in responses.

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.

The results are discussed in terms of DNA extract quality and quantity. DNA extract purity is of interests in terms of how contaminants will affect downstream assay performance. UV spectroscopy provides an indicator for different types of extract contaminants such as proteins, polysaccharides, and RNA [ 7 ]. PCR inhibition assays indicate whether contaminants will adversely affect the qPCR detection assay [ 29 ]. Purity ratios, obtained using UV spectroscopy, were outside the accepted range 1.

A high proportion of the S. Extraction methods are often optimized to enhance the removal of polysaccharides for example including the use of the surfactant ctyl trimethylammonium bromide or precipitation with high salt concentrations [ 36 , 37 ]. Similarly, the precipitation purification step included in three of the extraction methods reduced DNA contaminants as indicated by the higher proportion of extracts with purity ratios within the accepted range compared to the other extraction methods Figure 2.

For detection of pathogens in samples that are rich in polysaccharides it is essential to optimize the extraction method for their removal [ 30 , 37 ]. The primary limitation to UV spectroscopic DNA purity measurements is that the measurement only provides indicators for different types of contaminants and no information about the effect of these contaminants on downstream applications. Additionally, purity assays are sometimes used when specific contaminants are of interest, such as measuring UV absorbance of a sample at nm for humic acid detection [ 38 ].

A second limitation to the method is the required sample concentration, samples with measured concentrations less than PCR inhibitors are often associated with the sample matrix, including blood, food, water and soil [ 36 ].

For example, polysaccharides are known to interfere with downstream detection of plant diseases and pathogen contamination in food and water [ 36 ].

Although no inhibitors were expected because pure cultures were applied in this study, it is important to run inhibition assays to ensure the extracted purity is suitable for downstream applications. PCR inhibitors can lead to false negatives or underestimation of the quantity of a biological agent. Even though two different assays, one for inhibition and one for DNA quantification by qPCR, were used in this study and no inhibition was observed it is not likely that inhibitors were not detected as Qubit and qPCR measurements were in agreement Table 2.

Assays requiring larger target sequences are more likely adversely affected by shearing [ 27 , 28 ]. When DNA extracts are evaluated for shearing it is commonly performed using standard agarose gel electrophoresis [ 26 , 43 , 44 ].

The use of microfluidic gel electrophoresis allows for a lower limit of detection 11 ng per sample compared to 20 ng per band and semi-quantitative shearing analysis compared to standard gel electrophoresis [ 45 ]. The ideal target length for qPCR is 50 bp bp [ 46 ].

Overall 1. Slight shearing was observed for a number of the vegetative cell samples extracted with the bead beating method. Bead beating causes shearing but as observed in this study the degree of shearing varies and does not adversely affect most PCR applications using large quantities of genomic DNA [ 43 , 47 ]. 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 ].

While more accurate DNA concentration measurement methods are available including digital PCR and phosphorus elemental analysis [ 12 - 14 ] the methods used in this study represent those that are commonly used in molecular and diagnostic laboratories.

Each of the concentration methods used has different assumptions, and limitations. For example UV spectroscopy based concentration measurements requires two assumptions. First, DNA is the only molecule in the extract that absorbs light at nm and second, the DNA is all double-stranded. The UV spectroscopy DNA concentration measurements were statistically higher than the other two measurement methods Table 2. Absorbance based DNA concentration measurements commonly overestimate DNA concentrations for environmental samples due to contaminants from the sample matrix [ 48 ].

Accuracy of absorbance based DNA concentration measurements is dependent on sample purity [ 48 , 49 ]. The primary advantage of UV spectroscopy is the availability of microvolume instruments that are faster, easy to use, and requires less sample volume [ 48 ]. The Qubit and other bench top fluorometers allow for relatively fast DNA concentration measurements that are not as adversely affected by DNA contaminants as UV spectroscopy [ 45 ].

The accuracy of the fluorometric DNA concentration measurements is dependent on the accuracy of the DNA standards used [ 19 ]. Ideally the DNA concentration standard used should have a certified concentration that is traceable to the SI and has a published uncertainty value [ 19 ]. The standards in DNA concentration assay kits do not always meet this requirement and the concentration measurements should be evaluated with this in mind [ 50 ].

The advantage of using fluorometric DNA concentration measurement methods compared to qPCR is that no additional assay development is required for individual organisms.

DNA concentration measurements made using qPCR assume the number of within genome DNA sequence target copies is known and constant and that whole genomes are extracted [ 18 ]. The primary limitation to using qPCR for measuring DNA concentration is that; method development, validation, and execution are significantly more time intensive and costly than either of the two other methods.

However, qPCR is the only measurement method that measures the DNA concentration of a specific organism in a mixed sample e. The standard used can be a major source of qPCR measurement uncertainty [ 19 ].

No standard reference materials are available for the qPCR assays used in this study and the associated uncertainty of the standards used in this study was unknown. Finally, extraction efficiencies ranged by method and even varied for the three methods that used precipitation and silica spin columns for purification and bead beating for lysis indicating that extraction efficiency was dependent on more than the fundamental properties of the extraction methods.

It is important to note when discussing reported extraction efficiencies that quantity and quality are factors of the kits intended use as there is a tradeoff between the two. For example the magBeads extraction method was designed for use in field applications with limited resources and user experience. While the concentration of the resulting DNA was low, the extraction efficiency was comparable to the other extraction methods Table 3 , Figure 4. Similarly, the lysis step for the chemLysis method was optimized for Gram-negative bacteria as evident with the higher extraction efficiency for B.

Soils are rich in PCR inhibiting humic acids [ 36 ] and the precipS extraction method, optimized for extracting DNA from soils, included a purification step to remove humic acids. Additional, steps may have resulted in loss of genomic DNA.

A range of different size beads are used in the bead beating step of precipB which may aide in lysis of different cell types resulting in the statistically similar extraction efficiencies between all vegetative cell types not observed for the other extraction methods Figure 4.

Analysis of DNA yields using different size beads found that higher yields for hard to lyse Mycobacteria were obtained using smaller sized beads 0. For precipG quantity was sacrificed as the quantity of the intended sample type, microbial cultures, is not limited.

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.

When optimizing for true unknowns it is advantageous to use extraction methods that are optimal for hard-to-lyse cells. The limited number of cell types and extraction methods evaluated here were not intended to be exhaustive or to guide extraction method selection but rather to present the limitations and advantages of different extract characterization methods.

The use of only a single sample type was a limitation of the study and additional sample types would have better challenged the inhibition assay. The percentage of agarose in the gel will determine what size range of DNA will be resolved with the greatest clarity. Concentration and yield can be determined after gel electrophoresis is completed by comparing the sample DNA intensity to that of a DNA quantitation standard.

Standards used for quantitation should be labeled as such and be the same size as the sample DNA being analyzed. Because ethidium bromide is a known mutagen, precautions need to be taken for its proper use and disposal.

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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. Run DNA samples include several amounts ranging from 25 to ng on a 0. To maintain constant background staining of the gel, include 0. Use a UV light to photograph the gel. Compare fluorescence intensities and estimate DNA concentrations.

Also spot several DNA standards of known concentration. Let the gel stand for a few hours at room temperature to allow small contaminating molecules to diffuse away. Compare fluorescence intensities and estimate nucleic acid concentrations.

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