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.     

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.     

Comparison of three common DNA concentration measurement methods

Accurate measurement of DNA concentration is important for DNA-based biological applications. DNA concentration is usually determined by the ultraviolet (UV) absorption, fluorescence staining, and diphenylamine reaction methods. However, the best method for quality assurance of measurements is unknown. Here, we comprehensively compared these methods using different types of samples. We found that all three methods accurately determined the concentrations of high-purity DNA solutions. After digestion of DNA samples, concentration measurements revealed that the PicoGreen dye method was very sensitive to the degradation of DNA. The three methods displayed different anti-jamming ability when contaminants such as transfer RNA (tRNA), protein, and organic chemicals were included in DNA solutions. The diphenylamine reaction method gave the highest accuracy, with an average error of approximately 10% between measured and true values. The PicoGreen dye method was influenced by tRNA and protein, and the UV absorption method was susceptible to all kinds of impurities. Overall, the diphenylamine reaction method gave the most accurate results when DNA was mixed with contaminants, the PicoGreen dye method was most suitable for degraded DNA samples or DNA extracted from processed products, and the UV absorbance method was best for evaluating the impurities in DNA solutions.     

Fluorometric determination of adenine and its derivatives by reaction with glyoxal hydrate trimer

A fluorometric determination of adenine has been devised. It was found that adenine gives strong fluorescence when reacting with glyoxal hydrate trimer in an acidic medium. The maximum wavelengths of excitation and emission spectra were 328 and 382 mμ, respectively. This reaction was successfully applied to determination of 0.2–1.0 or 2.0–10.0 mμmoles of adenine and its derivatives, in which the hydrogen atom at position 9 is substituted with ribose or substituted ribose. Other nucleic acid bases, guanine, uracil, thymine, and cytosine, did not interfere with this fluorometric determination of adenine.     

Secondary structure of nucleic acids in the folded chromosome from E. coli.

The circular dichroism of membrane-free folded chromosomes from E. coli was measured and analyzed. The spectrum can be explained as a simple linear combination of the individual spectra of E. coli RNA, and E. Coli DNA in the B form. No contribution from A form or C form DNA was detected. There was evidently some real variation in the ratio of the two nucleic acids from preparation to preparation, but the average value was 24% RNA and 76% DNA. No significant light scattering was observed and the analyses indicated no contribution to the circular dichroism from scattering artifacts. Apparently, combining DNA, RNA, and protein into membrane-free folded chromosomes does not change the secondary structure of the DNA or RNA from that found for the free nucleic acid in the same solvent system.     

Optimization of acetonitrile co-solvent and copper stoichiometry for pseudo-ligandless click chemistry with nucleic acids

The copper(I) catalyzed azide-alkyne cycloaddition 'click' reaction yields a specific product under mild conditions and in some of the most chemically complex environments. This reaction has been used extensively to tag DNA, proteins, glycans and only recently RNA. Click reactions in aqueous buffer typically include a ligand for Cu(I), however we find that acetonitrile as a minor co-solvent can serve this role. Here we investigate the click labeling of RNA and DNA in aqueous buffer to determine the relationship between the stoichoimetry of Cu(I) and the acetonitrile co-solvent that affects nucleic acid stability. We find that very low concentrations of acetonitrile perform equally well and obviate the need for any additional Cu(I) stabilizing ligand. These pseudo-ligandless reaction conditions are optimal for nucleic acids click conjugations.     

Platelet adenylate cyclase and phospholipase C are affected differentially by ADP-ribosylation. Effects on thrombin-mediated responses.

During the isolation of the activator protein for glucosylceramide beta-glucosidase, we found that certain column fractions contained an inhibitor of the enzyme. After separation from the activator protein by a DEAE-Sephacel column, the inhibitor was purified further with a Spehadex G-75 column. The u.v. absorption spectrum of the purified material was similar to that of nucleic acids and the protein content of the purified material was negligible. Furthermore the purified inhibitor reacted with orcinol but not with diphenylamine, and its inhibitory activity was completely destroyed by treatment with RNAases. It seems likely that the purified inhibitor was tRNA. Authentic RNA, tRNA and DNA had similar inhibitory effects on beta-glucosidase (Ki 17 micrograms/ml for tRNA, noncompetitive toward the substrate). The inhibitory effect of nucleic acids was not fully overcome by an excess amount of the activator protein, but phosphatidylserine could restore the activity to normal. Tests with several other hydrolases revealed that the inhibitory effect of nucleic acids was fairly general.     

Spectrophotometric studies on the binding with polynucleotides of 4,4'-diacetyldiphenylurea-bis(guanylhydrazone) and methylglyoxal-bis(guanylhydrazone)

4,4'-Diacetyldiphenylurea-bis(guanylhydrazone) (DDUG), an agent very effective against several animal leukemias and tumors, was found, spectrophotometrically, to interact in a biphasic manner with several natural, native and heat-denatured, and synthetic DNAs. The spectrum of DDUG was shifted towards the visible region with a hypochromic shift reaching a maximum hypochromicity at 316 mμ at a 1: 1 molar ratio of DDUG to DNA nucleotide. Increasing molarity of DNA nucleotide resulted in a further shift towards the visible end, but with hyperchromicity rather than hypochromicity, and reaching its peak at 323 mμ. The interaction with yeast RNA was much weaker than that with DNA. 4,4'-Diacetyldiphenylurea (DDU) did not show any interaction with DNA; its monoguanylhydrazone (DDUM) showed only a hypochromic interaction. In contrast to DDUG, methyl-glyoxal-bis(guanylhydrazone) (CH₃-G), an aliphatic bisguanylhydrazone with antileukemic properties, showed only a hypochromic interaction with DNA at low ionic strength. Unlike DDUG, CH₃-G was a very weak inhibitor of the DNA polymerase reaction. The hypochromic shift of the DDUG spectrum with DNA was abolished in the presence of 15 mM sodium citrate or 500 mM NaCl but not in the presence of 150 mM NaCl or 100 mM sodium acetate. The hyperehromic shift was abolished in the presence of 8 M urea. From the results obtained with different DNAs, RNA, synthetic polynucleotides and nucleotides, it appears that the total shift of the DDUG spectrum in the presence of intact DNA can not be ascribed to interaction with a single base although a greater shift occurred in the presence of G-C rich DNA.     

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.     

THE NUCLEIC ACIDS OF BASAL BODIES ISOLATED FROM TETRAHYMENA PYRIFORMIS

Pellicular fragments were isolated from ethanol-fixed cells of the holotrichous ciliate Tetrahymena pyriformis by the action of digitonin. The isolated pellicles were further fragmented and the basal bodies of the cilia isolated from them by three methods. The preparations, examined in the electron microscope as embedded sections or negatively stained samples, consisted mainly of somewhat deformed pellicular material, the bulk of which was basal body. DNA was determined by the diphenylamine method and by reaction with DNase, and RNA, by the orcinol method. Nucleic acids were isolated by phenol extraction and analyzed spectrophotometrically and by reaction with RNase. The assays indicated 1.2 to 2.6 per cent RNA, similar to previously published work, but only 0.0 to 1.0 per cent DNA, near enough the sensitivity limits to render the presence of DNA in the preparations uncertain. Although the isolation procedure removed nuclear contents and ribosomes, the nucleic acids could still be a residual contaminant bound to the pellicle during the isolation. Hypotheses of basal body self-duplication, moreover, can be constructed both with and without nucleic acids.     

Pelagic-benthic coupling of nucleic acids in an abyssal location of the northeastern Atlantic Ocean.

Spatial and temporal changes in sedimentary nucleic acid concentrations in an abyssal locality of the northeastern Atlantic Ocean were investigated in relation to fluxes of nucleic acids produced in the photic layer. Sediment trap material, collected between 1996 and 1998 at depths of 1,000, 3,000, and 4,700 m, and sediment samples were analyzed for DNA and RNA content. Nucleic acid concentrations in the sediments were very high and displayed significant temporal changes, whereas mesoscale variability was low. DNA and RNA concentrations generally displayed opposite temporal patterns, which are likely to be dependent on the nature and characteristics of DNA and RNA molecules. Nucleic acid fluxes were high and displayed clear seasonal changes apparently coupled with seasonal pulses of primary production. However, while median values of DNA fluxes were relatively similar in all sediment traps, median values of RNA fluxes almost doubled from the 1,000- to the 4,700-m depth, suggesting differences in the metabolic activity of microbes associated with sinking particles. Significant relationships between DNA concentrations in the sediments and DNA fluxes and between RNA concentrations and RNA fluxes, indicating the presence of a clear pelagic-benthic coupling of particulate nucleic acids, were observed. The benthic system investigated was not steady state since we estimated that, from September 1996 to October 1998, nucleic acid concentration in the sediments decreased by about 165 mg of DNA m(-2). Vertical profiles revealed a significant decrease in DNA concentration with depth in the sediments, reaching an asymptotic value of about 5 microg g(-1). This DNA fraction constitutes a pool of potentially refractory DNA (accounting for 16 to 40% of the total DNA pool) that might be buried in the sediments.     

Escherichia coli topoisomerase III-catalyzed cleavage of RNA.

Relaxation of superhelical DNA by Escherichia coli topoisomerase III (Topo III) was inhibited by the inclusion of tRNA in the reaction mixture. Investigation of the basis of this inhibition revealed that Topo III could bind RNA and establish a cleavage-religation equilibrium. The addition of SDS to these reaction mixtures induced cleavage of the RNA by Topo III. The nucleotide sequences of RNA and DNA cleavage sites were identical, although cleavage site preference differed. Thus, the possibility that Topo III can pass strands of RNA as well as strands of DNA must be considered in accounting for the role of this enzyme in nucleic acid metabolism.     

Thermal difference spectra: a specific signature for nucleic acid structures

We show that nucleic acid structures may be conveniently and inexpensively characterized by their UV thermal difference spectra. A thermal difference spectrum (TDS) is obtained for a nucleic acid by simply recording the ultraviolet absorbance spectra of the unfolded and folded states at temperatures above and below its melting temperature (T_m). The difference between these two spectra is the TDS. The TDS has a specific shape that is unique for each type of nucleic acid structure, a conclusion that is based on a comparison of >900 spectra from 200 different sequences. The shape of the TDS reflects the subtleties of base stacking interactions that occur uniquely within each type of nucleic acid structure. TDS provides a simple, inexpensive and rapid method to obtain structural insight into nucleic acid structures, which is applicable to both DNA and RNA from short oligomers to polynucleotides. TDS complements circular dichroism as a tool for the structural characterization of nucleic acids in solution.     

Characterization of alfalfa-mosaic-virus protein polymerization in the presence of nucleic acid

The polymerization of alfalfa mosaic virus (AMV) protein in the presence of homologous nucleic acids and a number of other natural and synthetic nucleic acids was studied. The conditions for optimal assembly were found to be pH 6.0 and low ionic strength (I = 0.1 M) at room temperature, irrespective of the type of nucleic acid. The resulting nucleoprotein particles exhibited the same structural characteristics as the virus. This information emerged from optical diffraction and computer filtering of electron micrographs from the reconstituted particles. Irrespective of the type of nucleic acid present the polymerization of the protein resulting in a nucleoprotein particle is a cooperative process. Evidence for this was obtained by nitrocellulose filter binding assay, sodium dodecylsulphate/polyacrylamide gel electrophoresis, sedimentation velocity and electron microscopy of the reaction mixtures. The rates and efficiencies of reconstitution were of the same order of magnitude for a number of ribonucleic acids. Sedimentation data derived from AMV protein and AMV RNA mixtures suggested the existence of a specific nucleation product in the first stage of assembly. The results are discussed in terms of a tentative model of the assembly, in which at least two different steps (nucleation and elongation) can be distinguished, each characterized by an association constant.     

Tyrosine mutant helps define overlapping CD bands from fd gene 5 protein · nucleic acid complexes

We used a mutant gene 5 protein (g5p) to assign and interpret overlapping CD bands of protein · nucleic acid complexes. The analysis of overlapping protein and nucleic acid CD bands is a common challenge for CD spectroscopists, since both components of the complex may change upon binding. We have now been able to more confidently resolve the bands of nucleic acids complexed with the fd gene 5 protein by exploiting a mutant gene 5 protein that has an insignificant change in tyrosine optical activity at 229 nm upon binding to nucleic acids. We have studied the interactions of the mutant Y34F g5p (Tyr-34 substituted with phenylalanine) with poly[r(A)], poly[d(A)], and fd single-stranded DNA (ssDNA). Our results showed the following: (1) The 205–300 nm spectrum of poly[r(A)] saturated with the Y34F mutant (P/N = 0.25) was essentially the sum of the spectra of poly[r(A)] at a high temperature plus the spectrum of the free protein, except for a minor negative band at 257 nm. (2) The spectra of poly[d(A)] and fd ssDNA saturated with the mutant protein at a P/N = 0.25, minus the spectra of the free nucleic acids at a high temperature, also essentially equaled the spectrum of the free protein in the 205–245 nm region. (3) While the overall secondary structure of the Y34F protein did not change upon binding to any of these nucleic acids, there could be changes in the environment of individual aromatic residues. (4) Nucleic acids complexed with the g5p are unstacked (as if heated) and (in the cases of the DNAs) perturbed as if part of a dehydrated double-stranded DNA. (5) Difference spectra revealed regions of the spectrum specific for the particular nucleic acid, the protein, and whether g5p was bound to DNA or RNA. © 1997 John Wiley and Sons, Inc. Biopoly 42: 337–348, 1997     

Spectrophotometric analysis of RNA and DNA using cetyltrimethylammonium bromide

A versatile procedure is described for the analysis of RNA and DNA in brain using cetyltrimethylammonium bromide as the initial precipitant. Optimal conditions are described for the precipitation, hydrolysis, and effective separation of the RNA and DNA fractions from contaminating protein. The RNA and DNA fraction can now be accurately estimated by uv absorbance without a two wavelength correction. This method has also been used for the analysis of other mammalian organs and for mammalian cells obtained from tissue culture. The method may also be used for the simultaneous determination of radioactivity in nucleic acids. The orcinol reaction is shown to give high values for brain RNA.     

Steric structure of L-proline oligopeptides. II. Far-ultraviolet absorption spectra and optical rotations of L-proline oligopeptides

The results of the measurement of the far-ultraviolet absorption spectra of L -proline oligomers in water and acetonitrile are summarized as follows. The monomer has an absorption maximum at 182.5 mμ in acetonitrile. The absorption maximum of the dimer is found at 185 mμ and a shoulder appears around 200 mμ, that is, splitting of the absorption spectrum is observed in the dimer. As the degree of polymerization increases, the position of the shoulder shifts toward the wavelength of the absorption maximum of poly-L -proline II, with an accompanying increase in intensity. We may describe the absorption peak around 203 mμ of poly-L -proline II as identical with the shoulder with an increased intensity. By measurements of optical rotatory dispersion and circular dichroic spectra, it was also confirmed that the appearance of the helical conformation commences at the tetramer. When the number of residues is five or greater, the conformation of the helical structure of poly-L -proline II seems to be completed.