Factors influencing the extraction and determination of nucleic acids in plant material: Length of homogenization and precipitation from sodium chloride extracts

When studying the determination of nucleic acids in pollen based on extraction with hot 10% NaCl, further factors influencing the results were observed. Prolongation of the homogenization from 3 to 5 or 7 min caused considerable loss of DNA in extracts. On using trichloracetic acid to precipitate nucleic acids from the NaCl extracts, higher final values for the sum of nucleic acids were obtained than by precipitating with perchloric acid.     

Conditions for the extraction of DNA and RNA from tobacco pollen by sodium chloride and perchloric acid

Studying the influence of the pH of 10% NaCl solutions used for the extraction of RNA and DNA on the yield of both nucleic acids, the maxima of pH were found at which both types of nucleic acids pass into extracts better than at neutral pH and do not remain in residues of the experimental material.The perchloric acid extraction temperature was also studied for obtaining the hydrolysate of nucleic acids from the trichloroacetic acid precipitate of sodium chloride extracts differing by 5 °C within the range of 35 °C to 90 °C and it was found that in this wide range almost the same amount of RNA is extracted by the method used. However, at a lower temperature, some DNA remained in the extracted residue of the trichloroacetic acid precipitate of sodium chloride extracts.     

Use of acid,alkali and enzymes for the extraction of nucleic acids from pollen

Various methods for extracting nucleic acids from pollen were tested to find a suitable procedure for obtaining a pure preparation of nucleic acids uncontaminated by polysaccharides and polyphosphates without the use of ion exchangers. Extraction was carried out with perchloric acid, potassium hydroxide, ribonuclease and deoxyribonuclease, sodium tetraborate, and combinations of these. In all fractions, residues of precipitates and residues of extracted pollen matter, the quantity of RNA, DNA, proteins and concomitants,i.e. Polysaccharides and polyphosphates, was determined. The purity of preparations was checked by means of UV-spectra. The criterion of nucleic acid purity was agreement between the nucleic acid amounts calculated on the basis of measurement of absorption in UV-region, orcinol reaction and content of phosphorus. It was found that in our material only a few methods would be applicable and with great limitation, because many polysaccharides and polyphosphates appeared in nucleic acid fractions.     

Extracting nucleic acids from activated sludge which reflect community population diversity

Critical to most studies in molecular microbial ecology is the application of DNA/RNA extraction methods which can reveal the true level of population biodiversity present in samples from the community under investigation. Activated sludge communities have been studied extensively using molecular methods, but rarely have the nucleic acid isolation methods applied been assessed for their ability to achieve this. This study compares eight published RNA and DNA extraction protocols and one commercially available DNA isolation kit for their capacity to provide high quality nucleic acids that reflect the community composition. Each method was assessed on the basis of nucleic acid yield, purity and integrity, and the ability to provide PCR amplifiable RNA and DNA from known marker populations that varied in their resistance to nucleic acid extraction. Only three consistently provided DNA from each of the marker populations known to be present in the samples from fluorescence in situ hybridisation analysis. The failure of the other methods emphasises the need to validate all DNA/RNA extraction protocols. It is recommended that several validated extraction methods be used and the extracts pooled to further minimise any risk of bias.     

Altered nucleic acid partitioning during phenol extraction or silica adsorption by guanidinium and potassium salts

Nucleic acids were found to partition into the phenol phase during phenol extraction in the presence of guanidinium at certain concentrations under acidic conditions. The guanidinium-concentration-dependent nucleic acid partitioning patterns were analogous to those of the nucleic acid adsorption/partitioning onto silica mediated by guanidinium, which implied that phenol and silica interact with nucleic acids through similar mechanisms. A competition effect was observed in which the nucleic acids that had partitioned into the phenol phase or onto the silica solid phase could be recovered to the aqueous phases by potassium in a molecular weight–salt concentration-dependent manner (the higher molecular weight nucleic acids needed higher concentrations of potassium to be recovered, and vice versa). Methods were developed based on these findings to isolate total RNA from Escherichia coli. By controlling the concentrations of guanidinium and potassium salts used before phenol extraction or silica adsorption, we can selectively recover total RNA but not the high molecular weight genomic DNA in the aqueous phases. Genomic DNA-free total RNA obtained by our methods is suitable for RT-PCR or other purposes. The methods can also be adapted to isolate small RNAs or RNA in certain molecular weight ranges by changing the salt concentrations used.     

Improved preparation of lipoteichoic acids

A procedure is described for measuring the extraction of lipoteichoic acids from gram-positive bacteria in absolute terms. Virtually complete extraction was achieved from various bacteria by hot phenol/water if the cells were disrupted. Extraction of whole and delipidated cells and of the membrane fraction gave considerably lower yields. Most of the nucleic acids co-extracted from disrupted cells was removed by treatment with nucleases. Nuclease-resistant nucleic acid, protein, polysaccharide, and teichoic acid were separated from lipoteichoic acid by anionexchange chromatography on DEAE-Sephacel or hydrophobic interaction chromatography on octyl-Sepharose. Purified preparations were essentially free of polymeric contaminants, retained their alanine ester substitution, and were in the sodium salt form. Hydrophobic interaction chromatography also made it possible to recognize contamination of lipoteichoic acid with its deacylated and lyso-form, and to discriminate molecular species containing two and three, or two and four acyl groups.     

Improved protocol for high-quality Co-extraction of DNA and RNA from rumen digesta

We report an improved method for total nucleic acids extraction from rumen content samples. The method employs bead beating, and phenol-chloroform extraction followed by saline-alcohol precipitation. Total nucleic acids and RNA yield and purity were assessed by spectrophotometric measurements; RNA integrity was estimated using Agilent RNA 6000 Nano Kit on an Agilent 2100 Bioanalyzer. The method provided total nucleic acids and RNA extracts of good quantity and quality. The extraction is not time consuming and it is valuable for ecological studies of rumen microbial community structure and gene expression.     

14种蜂花粉的DNA和RNA分析

本文使用紫外光吸收法,分析了芝麻、葵花、泡桐等１４种蜂花粉中核酸（ＤＮＡ和ＲＮＡ）的含量,以进一步探讨花粉的抗衰老作用。结果说明不论那一种花粉,均含有丰富的核酸,但种类不同的花粉其中核酸的含量是不完全相同的。     

Alkylation of nucleic acid components by ethyleneimine derivatives. I. Alkylation of bases

In the course of investigating the reaction conditions of the nucleic acid components alcylation, the interaction of thioTEPA (N,N',N'-triethylenethiophosphoamide) with hydrochloric and perchloric acids was studied, perchloric acid increasing the alkylation products yield. HPLC and UV spectroscopy were used to isolate and identify products of nucleic bases alkylation by ethylenimine and its derivatives (thioTEPA and monoaziridinediethylphosphate). It is shown that under neutral conditions phosphoaminoethylation takes place, whereas under slightly acidic conditions products of aminoethylation are formed.     

Evaluation of extraction and purification methods for obtaining PCR-amplifiable DNA from compost for microbial community analysis

Analysis of microbial community structure in complex environmental samples using nucleic acid techniques requires efficient unbiased DNA extraction procedures; however, humic acids and other contaminants complicate the isolation of PCR-amplifiable DNA from compost and other organic-rich samples. In this study, combinations of DNA extraction and purification methods were compared based on DNA yield, humic acid contamination, PCR amplifiability, and microbial community structure assessed by terminal restriction fragment length polymorphisms (TRFLP) of amplified 16S rRNA genes. DNA yield and humic acid contamination, determined by A230, varied significantly between extraction methods. Humic acid contamination of DNA obtained from compost decreased with increasing salt concentration in the lysis buffer. DNA purified by gel permeation chromatography (Sepharose 4B columns) gave satisfactory PCR amplification with universal eubacterial 16S rRNA gene primers only when A260/A280 ratios exceeded 1.5. DNA purified with affinity chromatography (hydroxyapatite columns), and showing A260/A280 ratios as high as 1.8, did not show consistently satisfactory PCR amplification using the same 16S rRNA primers. Almost all DNA samples purified by agarose gel electrophoresis showed satisfactory PCR amplification. Principal components analysis (PCA) of TRFLP patterns differentiated compost types based on the presence/absence of peaks and on the height of the peaks, but differences in TRFLP patterns were not appreciable between extraction methods that yielded relatively pure DNA. High levels of humic acid contamination in extracted DNA resulted in TRFLP patterns that were not consistent and introduced a bias towards lower estimates of diversity.     

Determination of the ADP concentration available to participate in energy metabolism in an actin-rich cell, the platelet.

Almost all cells contain actin, which in its polymerized form, F-actin, binds 1 molecule of ADP/monomer. Little is known about the availability to metabolism of this bound ADP. A comparison was therefore made between perchloric acid and EDTA/ethanol extracts of human blood platelets. When the cells were extracted under conditions where the ATPase activity was negligible, the ethanol extracts had a 75% higher ATP/ADP ratio and a higher adenylate energy charge than perchloric acid extracts. The methods differed in that a considerable portion of protein-bound ADP was not extracted by ethanol. This bound ADP behaved as though it were unavailable to energy metabolism and should thus be considered as a compartment separate from the bulk metabolic pool of extragranular platelet adenine nucleotides. These results suggest that the level of ADP obtained with the common acid extraction overestimates the level available to participation in metabolism.     

Towards a universally adaptable method for quantitative extraction of high-purity nucleic acids from soil

A universally adaptable protocol for quantitative extraction of high-purity nucleic acids from soil is presented. A major problem regarding the extraction of nucleic acids from soil is the presence of humic substances, which interfere with the extraction process itself and in subsequent analytical manipulations. By the approach described here, the humic compounds are precipitated prior to cell lysis with Al(2)(SO(4))(3), and thus eliminated prior to the nucleic acid extraction. The protocol allows for removing of a considerable content and range of humic acids and should therefore be applicable for a wide spectrum of soil types. Accordingly, reproducible results in analyses of different soil types are made possible, inclusively for quantitative comparisons.     

Die Bestimmung der Nucleinsäuren inChlorella-Kulturen

Zusammenfassung Der Einfluß der Extraktionsbedingungen auf die Bestimmung der Nucleinsäuren beiChlorella pyrenoidosa wurde untersucht. Hohe Temperaturen und Perchlorsäure-Konzentrationen beschleunigten die Extraktion, verursachten aber gleichzeitig beträchtliche Verluste an, colorimetrisch mit der Indol- und Diphenylreaktion bestimmbaren DNS-Komponenten. Darüber hinaus enthielten diese Extrakte groß Mengen von Kohlenhydrat- und Proteinspaltprodukten, welche bei der UV-Absorption und der colorimetrischen Bestimmung von RNS und DNS störten.Zuverlässige Werte lieferte eine sechsstündige Extraktion des Zellmaterials bei 45°C mit 0,5 n Perchlorsäure und nachfolgende Bestimmung der RNS mit dem Orcintest und der DNS mit dem Diphenylamintest, ergänzt durch eine UV-Absorptionsmessung zur Kontrolle. Aus Polysacchariden stammende Glucose und Mannose verursachten einen Fehler bei der Orcinreaktion, der rechnerisch eliminiert werden konnte. Die Farbreaktionen zur DNA-Bestimmung zeigten sich empfindlich gegen hohe Perchlorsäure-Konzentrationen.DaChlorella große Mengen Polyphosphate enthält, führte die Nucleinsäurebestimmung auf der Basis des Phosphatgehaltes der Extrakte zu keinen brauchbaren Ergebnissen.

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Summary Effects of extraction conditions on the estimation of nucleic acids inChlorella pyrenoidosa were investigated. High temperatures and concentrations of perchloric acid accelerated the extraction, but they also caused appreciable losses of DNA components detectable colorimetrically with the indole and diphenylamine reaction. Moreover the extracts contained great amounts of breakdown products of carbohydrates and proteins, which interfered in the estimation of RNA and DNA based on colour reactions and UV-absorption.Reliable Results were obtained by extracting the cell material 6 hours at 45°C with 0,5 n perchloric acid and estimating RNA and DNA with the orcinol and diphenylamine reaction respectively, with an additional measurement of UV-absorption for control. The error in the orcinol reaction caused by mannose and glucose originating from polysaccharides could be eliminated arithmetically. Colour reactions for the estimation of DNA showed sensible against high concentrations of perchloric acid.Estimation of nucleic acids based on phosphorus content of extracts did not yield reliable results because of the high content of polyphosphates of the organism.

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Evaluation of commercial kits for extraction of DNA and RNA from Clostridium difficile

Commercial nucleic acid extraction kits are a cost effective, efficient and convenient way to isolate DNA and RNA from bacteria. Despite the increasing importance of the gastrointestinal pathogen, Clostridium difficile, and the increased use of nucleic acids in its identification, characterization, and investigation of virulence factors, no standardized or recommended methods for nucleic acid isolation exist. Here, we sought to evaluate 4 commercial DNA extraction kits and 3 commercial RNA extraction kits assessing cost, labor intensity, purity, quantity and quality of nucleic acid preparations. The DNA extraction kits produced a range of concentrations (20.9–546 ng/ml) and A_260/280 ratios (1.92–2.11). All kits were suitable for DNA extraction with the exception of the Roche MagNA pure LC DNA isolation kit III which produced DNA of high yield but with substantial shearing, but that did not affect downstream PCR amplifications. For RNA extraction, the Qiagen RNeasy mini kit stood out producing preparations of consistently higher concentrations and higher RNA integrity numbers (RIN). The Roche MagNA pure LC RNA isolation kit produced preparations that could not be properly assigned RINs due to a failure to remove small RNAs which were interpreted as degradation. Good DNA and RNA yield are critical but methods are often overlooked. This study highlights the potential for critical variation between established commercial systems and the need for assessment of any extraction methods that are used.     

Characterization of a Heat-Shock Process for Reduction of the Nucleic Acid Content of Candida utilis

A process for reducing the nucleic acid content of Candida utilis NRRL Y900 has been developed. The optimal process consists of heating the cells suspended in spent medium initially at pH 4.0 for various times at three different temperatures. Initially a heat-shock at 68 C for 1 to 3 sec is performed followed by incubation for 1 hr at 45 to 50 C and for a 2nd hr at 52 to 55 C. The distribution of degradation products has been characterized. Initially 90% of the nucleic acids were in a polymerized form (extractable by hot perchloric acid). After 30 min, much of this material was hydrolyzed but remained within the cell (extractable by cold perchloric acid). After 2 hr, most of the hydrolysis products leaked into the surrounding medium with only a small amount of low-molecular-weight material remaining within the membrane. Predominantly 3'-mononucleotides accumulated within the cell and eventually leaked from the cell.     

Ion-pairing high-performance liquid chromatographic method for the detection of N-acetylaspartate and N-acetylglutamate in cerebral tissue extracts

An ion-pairing high-performance liquid chromatographic method for the determination of N-acetylaspartate and N-acetylglutamate using a C-18 column and a UV detection at 210 nm wavelength, by means of a diode array detector, is presented. A buffer containing 2.8 mM tetrabutylammonium hydroxide, 25 mM KH(2)PO(4), 1.25% methanol, pH 7. 00, is utilized for the isocratic separation of these N-acetylated amino acids, at a flow rate of 1 ml/min and a column temperature of 23 degrees C. The suitability of this chromatographic separation (without additional chromatographic steps prior to HPLC assay) to monitor variations both of N-acetylaspartate and of N-acetylglutamate in perchloric acid brain extracts from rats subjected to the impact acceleration model of diffuse brain injury is also reported. According to the data presented, this HPLC method allows the separation of the two N-acetylated amino acids considered from the many possible interfering compounds, commonly present in extracts of cerebral tissue, which have high extinction coefficients at 210 nm wavelength. Values of N-acetylaspartate and N-acetylglutamate determined by this method showed that cerebral trauma negatively affects both compounds, according to the severity of trauma itself.     

Magnetic particles for the separation and purification of nucleic acids

Nucleic acid separation is an increasingly important tool for molecular biology. Before modern technologies could be used, nucleic acid separation had been a time- and work-consuming process based on several extraction and centrifugation steps, often limited by small yields and low purities of the separation products, and not suited for automation and up-scaling. During the last few years, specifically functionalised magnetic particles were developed. Together with an appropriate buffer system, they allow for the quick and efficient purification directly after their extraction from crude cell extracts. Centrifugation steps were avoided. In addition, the new approach provided for an easy automation of the entire process and the isolation of nucleic acids from larger sample volumes. This review describes traditional methods and methods based on magnetic particles for nucleic acid purification. The synthesis of a variety of magnetic particles is presented in more detail. Various suppliers of magnetic particles for nucleic acid separation as well as suppliers offering particle-based kits for a variety of different sample materials are listed. Furthermore, commercially available manual magnetic separators and automated systems for magnetic particle handling and liquid handling are mentioned.     

Evaluation of the efficiency of extraction of ultraviolet-absorbing pollen allergens and organic pollutants from airborne dust samples by capillary electromigration methods

Capillary electromigration methods, zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC), have been used for evaluation of the efficiency of different extraction agents applied to the extraction of pollen allergens and organic pollutants from dust samples collected during different periods (before, during and after pollen seasons) and in different locations in air-filtration devices (car-traffic tunnel in Prague and a metro station in Paris). Water and acetic acid extracts were analyzed by CZE using acetic acid as background electrolyte (BGE). Water and alkaline water-SDS-buffer extracts were analyzed by MEKC in Tris-phosphate BGE with anionic detergent sodium dodecylsulfate (SDS) micellar pseudophase. More material was extracted and more components were found in the water-buffer extracts than in the water extracts, and better resolution of the components was achieved by MEKC than by CZE. Significant differences have been found in the analyses of dust extracts of different origin. More material and more components have been found in the extracts of the dust collected in the pollen-rich period (March, April) than in the pollen-free period (December, January).     

Nucleic acid estimations in pollinated styles of Petunia hybrida

Summary The nucleic acid contents of styles with stigmas of Petunia hybrida have been estimated before and after pollination using a NaCl extraction procedure. No net changes in the amounts of nucleic acids are observed during pollen tube growth towards the ovary.