Effects of humic substances on fluorometric DNA quantification and DNA hybridization

DNA extracts from sediment and water samples are often contaminated with coextracted humic-like impurities. Estuarine humic substances and vascular plant extract were used to evaluate the effect of the presence of such impurities on DNA hybridization and quantification. The presence of humic substances and vascular plant extract interfered with the fluorometric measurement of DNA concentration using Hoechst dye H33258 and PicoGreen reagent. Quantification of DNA amended with humic substances (20-80 ng/microl) using the Hoechst dye assay was more reliable than with PicoGreen reagent. A simple procedure was developed to improve the accuracy for determining the DNA concentration in the presence of humic substances. In samples containing up to 80 ng/microl of humic acids, the fluorescence of the samples were measured twice: first without Hoechst dye to ascertain any fluorescence from impurities in the DNA sample, followed with Hoechst dye addition to obtain the total sample fluorescence. The fluorescence of the Hoechst dye-DNA complex was calculated by subtracting the fluorescence of the impurities from the fluorescence of the sample. Vascular plant extract and humic substances reduced the binding of DNA onto the nylon membrane. Low amounts (<2.0 microg) of humic substances derived from estuarine waters did not affect the binding of 100 ng of target DNA to nylon membranes. DNA samples containing 1.0 microg of humic substances performed well in DNA hybridizations with DIG-labeled oliogonucleotide and chromosomal probes. Therefore, we suggest that DNA samples containing low concentrations of humic substances (<20 ng/microl) could be used in quantitative membrane hybridization without further purification.     

Tandem immunoaffinity purification of protein complexes from Caenorhabditis elegans

The accurate determination of DNA concentration is essential for many processes in molecular biology and physiology and includes both gel- and cuvette-based methods. The recently introduced fluorescent dye, PicoGreen, has several advantages over other methods because it is sensitive and specific for double-stranded DNA (dsDNA). The dye is excited at 480 nm and emits at 520 nm when bound to dsDNA. This report describes the construction and use of PicoGmeter, a simple, inexpensive, fixed-wavelength fluorometer suitable for measuring PicoGreen fluorescence. PicoGmeter employs a blue light emitting diode (LED) for excitation and a photodiode to measure fluorescence. When compared to a commercially available instrument, PTI DeltaScan, the PicoGmeter performed admirably. Calibration curves for both instruments were superimposeable. Moreover, there was no significant difference between concentrations of DNA estimated by both instruments. A Bland and Altman analysis revealed that the PicoGmeter was equivalent to the PTI DeltaScan for estimating dsDNA concentration by the PicoGreen method. This simple, inexpensive, battery-operated fluorometer will allow investigators to employ the PicoGreen method without incurring the cost of purchasing a spectrofluorometer.     

Quantitation of DNA in buccal cell samples collected in epidemiological studies

Abstract

Buccal cell samples are increasingly used in epidemiological studies as a source of genomic DNA. The accurate and precise quantitation of human DNA is critical for the optimal use of these samples. However, it is complicated by the presence of bacterial DNA and wide inter-individual variation in DNA concentration from buccal cell collections. The paper evaluated the use of ultraviolet light (UV) spectroscopy, Höechst (H33258) and PicoGreen? as measures of total DNA, and real-time quantitative polymerase chain reaction (PCR) as a measure of human amplifiable DNA in buccal samples. Using serially diluted white blood cell DNA samples (at a concentration range of 300 to 0.5?ng µl^?1), UV spectroscopy showed the largest bias, followed by Höechst, especially for low concentrations. PicoGreen and real-time PCR provided the most accurate and precise estimates across the range of concentrations evaluated, although an increase in bias with decreasing concentrations was observed. The ratio of real-time PCR to PicoGreen provided a reasonable estimate of the percentage of human DNA in samples containing known mixtures of human and bacterial DNA. Quantification of buccal DNA from samples collected in a breast cancer case-control study by PicoGreen and real-time PCR indicated that cytobrush and mouthwash DNA samples contain similar percentages of human amplifiable DNA. Real-time PCR is recommended for the quantification of buccal cell DNA in epidemiological studies since it provides precise estimates of human amplifiable DNA across the wide range of DNA concentrations commonly observed in buccal cell DNA samples.     

Limitation of usage of PicoGreen dye in quantitative assays of double-stranded DNA in the presence of intercalating compounds

PicoGreen is a very sensitive fluorescent dye for quantitative assays of double-stranded DNA (dsDNA) in solution and is used in several analytical protocols in which sensitive and precise DNA detection is needed, also for examination of drug-DNA interactions. The data shown in this paper indicate that compounds intercalating to DNA influence the applicability of PicoGreen dye for quantitative measurements of dsDNA, and for this reason PicoGreen dye is not suitable for examination of drug-DNA interactions, especially interstrand DNA crosslinks.     

Comparison of DNA and RNA quantification methods suitable for parameter estimation in metabolic modeling of microorganisms

Recent developments in cellular and molecular biology require the accurate quantification of DNA and RNA in large numbers of samples at a sensitivity that enables determination on small quantities. In this study, five current methods for nucleic acid quantification were compared: (i) UV absorbance spectroscopy at 260 nm, (ii) colorimetric reaction with orcinol reagent, (iii) colorimetric reaction based on diphenylamine, (iv) fluorescence detection with Hoechst 33258 reagent, and (v) fluorescence detection with thiazole orange reagent. Genomic DNA of three different microbial species (with widely different G+C content) was used, as were two different types of yeast RNA and a mixture of equal quantities of DNA and RNA. We can conclude that for nucleic acid quantification, a standard curve with DNA of the microbial strain under study is the best reference. Fluorescence detection with Hoechst 33258 reagent is a sensitive and precise method for DNA quantification if the G+C content is less than 50%. In addition, this method allows quantification of very low levels of DNA (nanogram scale). Moreover, the samples can be crude cell extracts. Also, UV absorbance at 260 nm and fluorescence detection with thiazole orange reagent are sensitive methods for nucleic acid detection, but only if purified nucleic acids need to be measured.     

Use of diphenylamine as a colorimetric reagent for ribonucleic acid

RNA has been demonstrated to react with diphenylamine when acid hydrolysis is performed for 1 hour or more at 100°C. This reaction can be used for quantitative analysis of RNA, since there is a linear relationship between RNA concentration and absorbance. The reaction of RNA with diphenylamine can be quanlitatively distinguished from the reaction of DNA: the absorption spectrum of the RNA-diphenylamine reaction product has a maximum at 650 mμ, and a second, smaller peak at 490 mμ, while the DNA-diphenylamine reaction product has a single maximum at 605 mμ. It was found that, when mixtures of DNA and RNA are reacted with diphenylamine, the spectra reflect both the DNA:RNA ratios and the total amounts of nucleic acids. When the two-wavelength method of spectrum analysis was applied to such spectra, good agreement was found between actual and calculated values of nucleic acid concentrations. In this way, diphenylamine can be used for the simultaneous determination of the concentrations of DNA and RNA in mixtures. As is the case for the reaction of DNA with diphenylamine, it was found that the reaction of RNA is not altered by the presence of protein and that it involves primarily the purine nucleotides. The reaction of RNA with diphenylamine is discussed in relation to its possible analytical applications.     

Quantitation of adenovirus DNA and virus particles with the PicoGreen fluorescent Dye.

A microplate assay for the rapid quantitation of adenovirus DNA has been developed using the fluorescent dye PicoGreen, which selectively binds double-stranded DNA. The method was first applied to extracted adenoviral DNA and then extended to samples of intact, purified adenovirus after lysis of the viral capsid with the ionic detergent SDS. Utilizing the stoichiometric relationship between adenovirus DNA and intact particles, a physical particle count of intact virus is then derived for the sample. This PicoGreen-based assay has excellent reproducibility, linearity, and sensitivity. In its present form, this assay has a limit of quantitation of 10.3 ng/ml viral DNA, predicted to correspond to 2.6 x 10(8) virus particles/ml. This procedure was compared to a widely utilized spectroscopic method, in which samples are lysed with SDS and absorbance is read at 260 nm, and found to be 10- to 20-fold more sensitive. The dye binding assay also uses considerably less sample volume (<20%) than that needed for the spectroscopic method. Particle count values generated by the PicoGreen procedure are consistently lower (typically 1.5- to 2-fold) than this spectroscopic method. The applications and limitations of this method in the analysis of adenovirus samples are discussed.     

Fluorometric determination of deoxyribonuclease I activity with PicoGreen

A rapid and sensitive assay for the detection of deoxyribonuclease I (DNase I) activity is described. This method is based on the ability of PicoGreen dye to enhance its fluorescence when bound to double-stranded DNA. In the standard assay, reaction mixtures containing the DNase I sample and 0.2 microg of the substrate DNA were prepared in a fluorescence microtiter plate and incubated at 37 degrees C. At the end of the reaction, the diluted PicoGreen reagent was added to each well and fluorescence intensity was measured with a fluorescence plate reader. By this assay, it was possible to determine precisely as little as 5 pg of DNase I within an hour. Moreover, using a small amount of the substrate DNA, the method was shown to be suitable for the sensitive detection of DNase I inhibitor activity.     

Analysis of nucleic acids in Daphnia: development of methods and ontogenetic variations in RNA-DNA content

New sensitive fluorochromes, PicoGreen and RiboGreen (MolecularProbes, Inc.), were used to detect ng ml^-1 concentrations ofnucleic acids (RNA and DNA) in single Daphnia individuals ofall developmental stages. Nucleic acids were assayed by fluorescencewith a microplate reader using (i) RiboGreen (non-specific dye)and PicoGreen (DNA-specific dye) together and (ii) RiboGreenwith and without RNase. The two methods yield similar valuesfor DNA and RNA. The second method has the advantage of determiningboth RNA and DNA within a single sample aliquot, allowing maximumuse of available sample. In parthenogenically reproducing Daphniapulex, RNA and DNA concentrations were related to changes inmetabolic activity associated with moulting cycle and ontogeneticdevelopment. In juveniles, elevated DNA content was observedin the early postmoult, followed by an increase in RNA duringintermoult and premoult. RNA concentration peaked at the lateststages of embryonic development, followed by a gradual declineduring juvenile development, with the lowest values in adultfemales (eggs removed). Similarly, DNA concentrations were highestduring the early phase of postnatal development, decreasingwhen body size increased. Our results suggest that ontogeneticvariations have implications for the use of nucleic acids asa measure for growth in zooplankton and may provide useful insightsinto mechanisms of growth on the cellular level.     

The estimation of deoxyribonucleic acid in the presence of sialic acid: application to analysis of human gastric washings

1. Sialic acid has been found to interfere with three colorimetric reactions used for the estimation of DNA: a modified diphenylamine reaction at 100° (Dische, 1930), the nitrophenylhydrazine method (Webb & Levy, 1955) and the diphenylamine reaction at 30° (Burton, 1956). 2. Evidence is presented that sialic acid is present in hydrolysates obtained from gastric wash-out material. 3. A mathematical method for correcting for interference from sialic acid in the diphenylamine reaction at 30° is described. 4. The diphenylamine reaction has been modified to make it suitable for the estimation of DNA in the presence of sialic acid. The modifications are to increase the concentration of diphenylamine to 2% and to perform the reaction at 6–13° for 48hr. These modifications increase the sensitivity 25% above Burton's (1956) modification of the diphenylamine reaction. 5. The precipitation, extraction and recovery of DNA from gastric wash-out material have been investigated.     

Characterization of PicoGreen interaction with dsDNA and the origin of its fluorescence enhancement upon binding

PicoGreen is a fluorescent probe that binds dsDNA and forms a highly luminescent complex when compared to the free dye in solution. This unique probe is widely used in DNA quantitation assays but has limited application in biophysical analysis of DNA and DNA-protein systems due to limited knowledge pertaining to its physical properties and characteristics of DNA binding. Here we have investigated PicoGreen binding to DNA to reveal the origin and mode of PicoGreen/DNA interactions, in particular the role of electrostatic and nonelectrostatic interactions in formation of the complex, as well as demonstrating minor groove binding specificity. Analysis of the fluorescence properties of free PicoGreen, the diffusion properties of PG/DNA complexes, and the excited-state lifetime changes upon DNA binding and change in solvent polarity, as well as the viscosity, reveal that quenching of PicoGreen in the free state results from its intramolecular dynamic fluctuations. On binding to DNA, intercalation and electrostatic interactions immobilize the dye molecule, resulting in a >1000-fold enhancement in its fluorescence. Based on the results of this study, a model of PicoGreen/DNA complex formation is proposed.     

Measurements of DNA and RNA in mammary gland homogenates by the ethidium bromide technique.

A rapid method of determining simultaneously DNA and RNA in mammary gland homogenates using the ethidium bromide technique is discussed. The method utilizes a quantitative extraction of DNA and RNA with 2.0m sodium chloride, SDS, and EDTA at pH 8.0. Assays of mammary gland RNA and DNA using previously published methods were compared with determinations using the ethidium bromide technique. While the fluorescence method gave lower values for RNA when compared to those obtained using the orcinol or absorbancy ratio (OD 260nm/280nm), DNA measurements agreed well with the values determined by the diphenylamine technique. Extinction coefficient data for total mammary gland RNA isolated using a modified phenol extraction procedure are also presented.     

Application of sonication to release DNA from Bacillus cereus for quantitative detection by real-time PCR

A rapid sonication method for lysis of Gram-positive bacteria was evaluated for use in combination with quantitative real-time polymerase chain reaction (PCR) analyses for detection. Other criteria used for evaluation of lysis were microscopic cell count, colony forming units (cfu), optical density at 600 nm and total yield of DNA measured by PicoGreen fluorescence. The aim of this study was complete disruption of cellular structures and release of DNA without the need for lysing reagents and time-consuming sample preparation. The Gram-positive bacterium Bacillus cereus was used as a model organism for Gram-positive bacteria. It was demonstrated by real-time PCR that maximum yield of DNA was obtained after 3 to 5 min of sonication. The yield of DNA was affected by culture age and the cells from a 4-h-old culture in the exponential phase of growth gave a higher yield of DNA after 5 min of sonication than a 24-h-old culture in the stationary phase of growth. The 4-h-old culture was also more sensitive for lysis caused by heating. The maximum yield of DNA, evaluated by real-time PCR, from a culture of the Gram-negative bacterium Escherichia coli, was obtained after 20 s of sonication. However, the yield of target DNA from E. coli rapidly decreased after 50 s of sonication due to degradation of DNA. Plate counting (cfu), microscopic counting and absorbance at 600 nm showed that the number of viable and structurally intact B. cereus cells decreased rapidly with sonication time, whereas the yield of DNA increased as shown by PicoGreen fluorescence and real-time PCR. The present results indicate that 3-5 min of sonication is sufficient for lysis and release of DNA from samples of Gram-positive bacteria.     

Nucleic Acid (DNA, RNA) Quantification and RNA/DNA Ratio Determination in Marine Sediments: Comparison of Spectrophotometric, Fluorometric, and HighPerformance Liquid Chromatography Methods and Estimation of Detrital DNA

In this study, we compared three methods for extraction and quantification of RNA and DNA from marine sediments: (i) a spectrophotometric method using the diphenylamine assay; (ii) a fluorometric method utilizing selective fluorochromes (thiazole orange for total nucleic acids and Hoechst 33258 for DNA); and (iii) a high-pressure liquid chromatography (HPLC) method which uses a specific column to separate RNA and DNA and UV absorption of the nucleic acids for quantification. Sediment samples were collected in the oligotrophic Cretan Sea (eastern Mediterranean, from 40 to 1,540 m in depth) and compared to the distribution and composition of the benthic microbial assemblages (i.e., bacteria and microprotozoa). DNA concentrations measured spectrophotometrically and by HPLC were not significantly different, while fluorometric yields were significantly lower. Such differences appear mainly due to fact that the stain-DNA complex is strongly dependent on the DNA composition and structure. RNA concentrations determined by the three methods displayed some differences; fluorometric and spectrophotometric methods obtain RNA concentration by difference and therefore may be biased by DNA estimates. By contrast, the HPLC method provides independent assessments of RNA and DNA concentrations. We tentatively estimated the contribution of the detrital DNA to the total DNA pools in two ways. The two calculations provided quite similar results indicating that the majority of the DNA pool in the deep-sea sediments was detrital. Microbial RNA generally accounted for almost the entire sedimentary RNA pools below 100-m depth. RNA concentrations were found to decrease along the Cretan shelf and slope. The RNA/DNA ratio calculated by using fluorometric DNA concentrations was significantly correlated with values of sediment community oxygen consumption only below 100-m depth (dominated by the microbial biomass). These data suggest that the RNA/DNA ratio, based on fluorometric estimates of DNA, can be used as an indicator of benthic metabolic activity, but only when metazoan contribution to the microbial DNA is negligible.     

Extraction of nucleic acids from lyophilized plant material

Four methods for extracting nucleic acids from lyophilized cotton (Gossypium hirsutum L. cv. Stoneville 62) leaves and roots were compared. They were based on the use of: (I) HC10₄; (II) KOH; (III) a mixture of 90% phenol, Tris (hydroxymethyl) aminomethane buffer, and sodium lauryl sulfate; and (IV) NaCl. (I) extracted large amounts of RNA but little DNA and extracted much carbohydrate and protein contaminants. (II) gave a good yield of both RNA and DNA but extracted such large amounts of contaminating material that purification of RNA on an anion exchange column was necessary. (III) extracted only part of the RNA and practically no DNA, but extracted contaminating materials. (IV) resulted in high yields of both RNA and DNA when modified to omit preliminary acid extraction of impurities. The use of cold trichloroacetic acid instead of ethanol, to precipitate NaCl-extracted nucleic acids, separated the nucleic acids from most of the carbohydrate and acid-soluble phosphate contaminants and resulted in good agreement among results by ultraviolet absorbance, pentose tests, and phosphate analysis. This method also resulted in lower protein contents and better ultraviolet absorption spectra than the other methods tested. Nucleic acids were extracted from leaves of 14 other species of plants, in addition to cotton, by this modified NaCl procedure.     

Effects of chemistry, manufacturing and concentration of the dye basic fuchsin regarding its use in quantitative cytophotometry

Four separately manufactured preparations of basic fuchsin were compared to determine the effects of concentration, chemistry, and the manufacturing process for their quantitative value in the nuclear Feulgen reaction. In order to make the necessary comparisons, the two wavelength method of quantitative cytophotometry was employed to analyse each stain application regarding DNA measurements. Chicken erythrocytes, and myxamoebae and plasmodia of Didymium iridis were employed as experimental tissues. Results indicated that all four preparations of the stains yielded acceptable and valid quantitative data in relative DNA values. Differing manufacturers and dye concentrations had no appreciable effect on relative quantitative measurements. However, the maximum staining intensity was affected by differences both in the chemical structure of the individual stains and by the products from various manufacturers. The maximum dye intensity and accurate quantitative absolute values for DNA measurement were best obtained by the use of basic fuchsin having the same colour index (Cl) 42510 as that manufactured by Fisher Scientific Incorporated.     

OPTIMAL PURIFICATION AND SENSITIVE QUANTIFICATION OF DNA FROM FECAL SAMPLES

Application of reliable, rapid and sensitive methods to laboratory diagnosis of zoonotic infections continues to challenge microbiological laboratories. The recovery of DNA from a swine fecal sample and a bacterial culture extracted by a conventional phenol-chloroform extraction method was compared to a rapid silica-membrane spin-column method (DNeasy Tissue or QIAamp Stool Kit, QIAGEN GmbH). The two spin column methods yielded 3.5 and 2.7 μg of DNA, respectively, when the elution volume was 200 μL, compared to 1.3 and 1.5 μg of DNA, respectively, with the phenol-chloroform method.
In addition, the detection range of λ-DNA of a spectrophotometric and a fluorometric (PicoGreen) method was compared. The PicoGreen showed a quantification limit of 1 ng/mL, consistent triplicate measurements, and finally a linear relationship between the concentrations of DNA standards and the fluorescence readings (R²= 0.99 and R²= 1.00).
In conclusion, silica-membrane columns can provide a more convenient and less hazardous alternative to the conventional phenol-based method. The results have implication for further improvement of sensitive amplification methods for laboratory diagnosis.     

A comparison of rapid electrophoretic and chromatographic methods for the investigation of common histological dyes

Summary The compositions of fifty-nine common histological dyes, as well as duplicate samples of several dyes from different suppliers, have been studied by agar gel electrophoresis, agarose gel electrophoresis, paper electrophoresis, paper chromatography and thin layer chromatography. Tables are presented to show the number of components present in each dye as disclosed by the different methods; the cases where duplicate samples were available are summarised in a separate table.On the basis of effectiveness and convenience agar gel electrophoresis and thin layer chromatography were by far the best methods. The Chromatographic method was of slightly wider applicability but as electrophoretic methods gave information on dye charge, agar gel electrophoresis was the best single method.     

Development of a fast and reliable method for the assessment of microbial colonization and growth on textiles by DNA quantification

There is a lack of relevant methods to assess the colonization of textiles by skin bacteria because present methods are mainly culture-based procedures. Therefore, the goal of this study was to develop a fast and sensitive culture-independent procedure for the quantification of microbial colonization and growth on textiles. We have established a suitable protocol to use DNA quantification as a reliable method for in vitroand in vivoinvestigations of textiles. For DNA extraction, a two-step procedure comprising treatment of the textile with a solution containing Triton X-100 and lysozyme for 1 h and a successive treatment by SDS and proteinase K for 2 h turned out to be most efficient. DNA extracted from textiles and fabrics was than quantified with the highly sensitive PicoGreen fluorescent dye. In vitrochallenge tests demonstrated a strong correlation between numbers of bacteria on textiles and amount of DNA extracted from textiles. Therefore, this method was used to compare different materials after in vivotrials for assessment of their susceptibility for microbial colonization and growth.     

LAMP for human African trypanosomiasis: a comparative study of detection formats

Loop-mediated isothermal amplification (LAMP) is at the forefront of the search for innovative diagnostics for human African trypanosomiasis (HAT). Several simple endpoint detection methods have been developed for LAMP and here we compare four of these: (i) visualization of turbidity; (ii) addition of hydroxynaphthol blue before incubation; (iii) addition of calcein with MnCl₂ before incubation and (iv) addition of Quant-iT PicoGreen after incubation. These four methods were applied to four LAMP assays for the detection of human African trypanosomiasis, including two Trypanozoon specific and two Trypanosoma brucei rhodesiense specific reactions using DNA extracted from cryo-preserved procyclic form T. b. rhodesiense. A multi-observer study was performed to assess inter-observer reliability of two of these methods: hydroxynapthol blue and calcein with MnCl₂, using DNA prepared from blood samples stored on Whatman FTA cards. Results showed that hydroxynaphthol blue was the best of the compared methods for easy, inexpensive, accurate and reliable interpretation of LAMP assays for HAT. Hydroxynapthol blue generates a violet to sky blue colour change that was easy to see and was consistently interpreted by independent observers. Visible turbidity detection is not possible for all currently available HAT LAMP reactions; Quant-iT PicoGreen is expensive and addition of calcein with MnCl₂ adversely affects reaction sensitivity and was unpopular with several observers.