Dissolved oxygen alteration of the spectrophotometric analysis and quantification of nucleic acid solutions (original) (raw)
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UV absorbance spectrophotometry is widely used for the quantification of nucleic acids. For accurate quantification, it is important to determine the hypochromicity of the oligonucleotide or complex nucleic acid structure. The use of thermal denaturation studies in conjunction with UV spectrophotometry to determine hypochromicity requires prolonged, elevated temperatures, which may cause partial hydrolysis of RNA. In addition, dsRNA is difficult to denature even at elevated temperature, and the extinction coefficients of nucleic acids are also affected by temperature, which makes it difficult to accurately determine the nucleic acid concentration. To overcome these caveats, we have utilized the chemical denaturant dimethyl sulfoxide which, in conjunction with a short thermal denaturation, prevents renaturation of the duplex nucleic acids (dsDNA/ RNA). Using this approach, we have measured the absorbance of both the unstructured and structured nucleic acids to accurately measure their hypochromicity and determine their extinction coefficients. For a range of different dsRNA, we have for the first time determined values of 46.18−47.29 μg/mL/A 260 for the quantification of dsRNA using UV spectrophotometry. Moreover, this approach enables the accurate determination of the relative proportion of duplex nucleic acids in mixed ds/ss nucleic acid solutions, demonstrating significant advantages over current methods.
Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity
BioTechniques, 1997
The ratio of absorbance at 260 and 280 nm (the A260/280 ratio) is frequently used to assess the purity of RNA and DNA preparations. Data presented in this report demonstrate significant variability in the RNA A260/280 ratio when different sources of water were used to perform the spectrophotometric determinations. Adjusting the pH of water used for spectrophotometric analysis from approximately 5.4 to a slightly alkaline pH of 7.5-8.5 significantly increased RNA A260/280 ratios from approximately 1.5 to 2.0. Our studies revealed that changes in both the pH and ionic strength of the spectrophotometric solution influenced the A260/280 ratios. In addition, the ability to detect protein contamination was significantly improved when RNA was spectrophotometrically analyzed in an alkaline solution. UV spectral scans showed that the 260-nm RNA absorbance maximum observed in water was shifted by 2 nm to a lower wavelength when determinations were carried out in Na2HPO4 buffer at a pH of 8.5....
The measurement and distribution of dissolved nucleic acids in aquatic environments
Limnology and Oceanography, 1989
Nucleic acids (DNA and RNA) are ubiquitous components of the dissolved organic matter (DOM) pool of all oceanic, neritic, estuarine, and freshwater habitats studied to date. A new method for the quantitative determination ofdissolved nucleic acids (DNA and RNA) in water and scdimcnt samples was developed, evaluated, and utilized in a study of various marine and freshwater ecosystems. Under appropriate reaction conditions, dissolved DNA (D-DNA) and dissolved RNA (D-RNA) are efficiently removed from solution with the addition of cctyltrimethylammonium bromide (CTAB) and subsequent formation of insoluble CTA-nucleic acid salts. The insoluble salts are collected, by filtration, onto glass-fiber filters and analyzed for DNA and RNA with fluorometric and calorimetric procedures, respectively. The pcrformancc of this CTAB method is simple, reliable, and reproducible for measuring dissolved nucleic acids in natural aquatic environments. For the ecosystems investigated hcrcin, D-DNA and D-RNA concentrations ranged from 0.56 to 88 pg liter-' and 4.03 to 871 pg liter I; the ratio of D-RNA to D-DNA ranged from 4.1 to 11.5.
Comments on the determination of nucleic acids in natural waters by the CTAB-DABA-orcinol method
Science of The Total Environment, 1996
Karl and Bailiff (1989) presented the CTAB-DABA-orcinol method for the determination of dissolved DNA (D-DNA) and RNA (D-RNA) in natural waters. Here we demonstrate that, by this method, not only RNA but also heteropolysaccharides containing pentose sugars are precipitated and measured. RNase treatment resulted in only a slight reduction of the measured D-RNA concentrations in seawater. These findings indicate that the CTAB-orcinol method is not specific for measuring D-RNA. We improved the accuracy of the CTAB-DABA assay for determining D-DNA in seawater and we further show that viral DNA is probably also measured by the CTAB-DABA method.
Free Radical Biology and Medicine, 1999
Recently, an artifactual formation of a number of modified DNA bases has been alleged during derivatization of DNA hydrolysates to be analyzed by gas chromatography-mass spectrometry (GC-MS). These modified bases were 8-hydroxyguanine (8-OH-Gua), 5-hydroxycytosine (5-OH-Cyt), 8-hydroxyadenine (8-OH-Ade), 5-hydroxymethyluracil (5-OHMeUra), and 5-formyluracil, which represent only a small percentage of more than 20 modified DNA bases that can be analyzed by GC-MS. However, relevant papers reporting the levels of these modified bases in DNA of various sources have not been cited, and differences in experimental procedures have not been discussed. We investigated the levels of modified bases in calf thymus DNA by GC-MS using derivatization at three different temperatures. The results obtained with GC/isotope-dilution MS showed that the levels of 5-OH-Cyt, 8-OH-Ade, 5-OH-Ura, and 5-OHMeUra were not affected by increasing the derivatization temperature from 23°C to 120°C. The level of 8-OH-Gua was found to be higher at 120°C. However, this level was much lower than those reported previously. Formamidopyrimidines were readily analyzed in contrast to some recent claims. The addition of trifluoroacetic acid (TFA) adversely affected the levels of pyrimidine-derived lesions, suggesting that TFA is not suitable for simultaneous measurement of both pyrimidine-and purine-derived lesions. The data obtained were also compared with those previously published. Our data and this comparison indicate that no artifactual formation of 5-OH-Cyt, 8-OH-Ade, and 5-OHMeUra occurred under our experimental conditions in contrast to recent claims, and no prepurification of DNA hydrolysates by a tedious procedure is necessary for accurate quantification of these compounds. The artifactual formation of 8-OH-Gua can be eliminated by derivatization at room temperature for at least 2 h, without the use of TFA. The results in this article and their comparison with published data indicate that different results may be obtained in different laboratories using different experimental conditions. The data obtained in various laboratories should be compared by discussing all relevant published data and scientific facts, including differences between experimental conditions used in different laboratories.
Artifacts Associated with the Measurement of Oxidized DNA Bases
Environmental Health Perspectives, 1997
In this paper we review recent aspects of the measurement of oxidized DNA bases, currently a matter of debate. There has long been an interest in the determination of the level of oxidized bases in celiular DNA under both normal and oxidative stress conditionas. In this respect, the situation is confusing because variations that may be as large as two orders of magtude ba been reported for the yield of the formation of 8-oxo-7,8-dihydroguanine (8-oxoGua) in similar DNA samples. However, recent findings dearly show that application of several assays like gas chromatography-mass spectrometry (GC-MS) and [32P]-postlabeling may lead to a significant overestimation of the level ofoxidized bases in cellular DNA. In particular, the siblation step, which is required to make the samples volatile for the GC-MS analysis, has been shown to induce oxidation of normal bases at the level of about one oxidized base per 104 normal bases. This has been found to be a general process that applies in particular to 8-oxoGua, 8-oxo-7,8-dihydroadenine, 5-hydroxycytosine, 5-(hydroxymethyl)uracil, and 5-formyluracil. Interestingly, prepurification of the oxidized bases fiom DNA hydolysate prior to the derivatization reaction prevents arfactual oxidation. Under these conditions, the level of oxidized bases measured by GC-MS is similar to that obtained by HPLC associated with electrochemical detection (HPLC-EC). It should be added that the level of 8-oxo-7,8-dihydro-2'-doxynosine inm control cellular DNA has been found to be about fivefold lower than in earlier HPLC-EC measurements by using appropiate conditions of extraction and enzymatic digestion of DNA. Similar conclusions were reached by measuring formamidopyrimidine-DNA glycosylase sensitive sites as revealed by the single cel gel electrophoresis (comet) assay. Key words DNA base damage, DNA repair enzymes, oxidative lesions.
Determination of dissolved nucleic acids in seawater by the fluorescence dye, ethidium bromide
Marine Chemistry, 1992
A new method for the determination of dissolved double-stranded deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) in seawater was developed, evaluated and used to study the fates of these nucleic acids in marine ecosystems. These nucleic acids, which were pre-concentrated on a hydroxyapatite column, were determined fluorometrically by the use of ethidium bromide dye, which binds specifically to the double-stranded polynucleotide. No dissolved organic matter coexisting in the preconcentrated sample solution interfered in the analysis of DNA and RNA. Column recoveries of DNA and RNA in a sample volume of up to 11 were 93% and 97%, respectively, and 90% of both at 51. The detection limits of DNA and RNA concentrated from a 5 1 sample by this fluorometric method were 0.6 and 1.1/tg 1-1, respectively. The concentration of dissolved nucleic acids in the waters from Tokyo Bay and Sagami Bay showed great variation in space and time. DNA ranged from 1 to 32 #g 1-1, and RNA from below the detection limit to 34/tg 1-I. The total amount of phosphorus in nucleic acids was an important fraction (12.9 + 8.2%) of the dissolved organic phosphorus (DOP) and showed a good correlation with DOP.
Fluorometric quantification of RNA and DNA in solutions containing both nucleic acids
Analytical Biochemistry, 2003
A fluorescence-based method for quantitative determination of RNA and DNA in probes containing both nucleic acids has been developed. The total concentration of nucleic acids is determined using SYBR Green II dye under conditions providing independent binding of the fluorophore with DNA and RNA. The concentration of DNA is specifically measured using the Hoechst 33258 dye and the RNA concentration is calculated from these data. The procedure allows for accurate determination of DNA concentration in the range 10-1000 ng/ml in the presence of 200-fold excess of RNA and determination of RNA concentrations in the range 10-1000 ng/ml in the presence of large excess of DNA. An absence of the treatment of mixed samples with RNase-free DNase I provides rapid, reproducible, and accurate RNA quantification.