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.     

The effects of pH and salts on nucleic acid partitioning during phenol extraction

The partitioning of nucleic acids is sensitive to pH during phenol extraction. However, the exact effects of pH on phenol extraction had not been systematically investigated, and the mechanism of which were not fully elucidated. In this paper, we showed that the partitioning of nucleic acids was determined neither solely by the pH of the aqueous buffer being used, nor by the “pH of the phenol”; the latter is a completely wrong conception. We demonstrated that a key determinant for nucleic acid partitioning during phenol extraction was the equilibrated pH of the aqueous phase, which should be defined as the pH of phenol extraction. For example, when 50?mM NaAc-HAc buffer at pH of 3.47 was mixed with an equal volume of water-saturated phenol, the equilibrated pH of aqueous phase would be raised to ～3.84. At this pH, almost all of genomic DNA partitioned into the phenol phase, and genomic DNA-free total RNA was retained in the aqueous phase. Several salts were found affecting the partitioning of nucleic acids during phenol extraction in different manners. Based on these results, a low-cost and efficient method for genomic DNA-free total RNA extraction was developed.     

Light Effects on the Nucleic Acids of Excised Cotton Cotyledons

The effects of light and glucose in the nutrient medium on the nucleic acid metabolism of excised 8-day cotton (Gossypium hirsutum var. Acala 44) cotyledons were determined. The rates of synthesis as affected by light and glucose were determined by brief exposures to C(14)-labeled orotic acid. The nucleic acids were fractionated by homogenizing in Tris-HCl buffer and centrifuging to obtain soluble and microsomal RNA (20,000 x g supernatant) and a particulate nucleic acid fraction (20,000 x g precipitate) or by extracting in phenol followed by 10% NaCl extraction at 100 degrees . The phenol extract was analyzed by density gradient centrifugation.Light and glucose caused parallel changes in nucleic acid levels of the various fractions, in orotic acid-6-C(14) absorption and in rates of synthesis of nucleic acids. Light and glucose appear to enhance binding of the ribosome nucleic acid so that it becomes less extractable in Tris-HCl buffer or phenol. The bound nucleic acids were labeled at a slightly higher rate than the total nucleic acids extracted by Tris-HCl or phenol. However, light treatment for 48 hours promoted a very high labeling rate in the soluble, low molecular weight nucleic acid as shown by density gradient centrifugation of the phenol extractable fraction.It was concluded that a part of the nucleic acid changes were brought about by light acting through the photosynthetic production of carbohydrate. This conclusion was strengthened by the observation that herbicide inhibitors of photosynthesis and limited atmospheric CO(2) concentrations partially inhibited the nucleic acid changes. However, glucose did not cause changes in nucleic acid levels as large as those caused by light and changes were observed to occur in light even though the endogenous sugar levels were maintained at a low level by the inhibition of photosynthesis with herbicides. The data indicated that light may produce changes in nucleic acid levels by other mechanisms additional to those regulating the sugar level in the tissue.     

Comparison of Various Procedures for Removing Proteins and Nucleic Acids from Cell Walls of Bacillus Subtilis

Several procedures were used i n an attempt to prepare clean cell walls from Bacillus subtilis. The results indicate that protein and nucleic acids are tightly bound tothe walls. cleanest wall preparations were found following trichloroacetic acid extraction at 60° or by extraction with 0.lN NaOH under a nitrogen atmosphere for 10 hrs. Protein denaturants, such as sodium dodecyl sulfate and concentrated guanidine hydrochloride were relatively ineffective in removing proteins and nucleic acids from the cell walls. Cell wall-bound DNA was biologically The active i n transformation assays.     

Deproteination of nucleic acids by filtration through a hydrophobic membrane.

To remove or inactivate an enzyme from DNA in multistep procedures in molecular biology, it is often necessary to phenol extract the solution, followed by chloroform extraction and ethanol precipitation. In addition to being time-consuming and hazardous, there can be significant loss of DNA with this procedure, especially when small volumes or amounts of DNA are being used. We have found that filtering analytical reaction mixtures through a hydrophobic membrane specifically to remove protein is a rapid alternative to phenol extraction. Within broad limits commonly encountered in molecular biology, filtration through a polyvinylidene difluoride membrane quantitatively removes a variety of enzymes without significant loss of double-stranded nucleic acid.     

A robust,cost‐effective method for DNA,RNA and protein co‐extraction from soil,other complex microbiomes and pure cultures

The soil microbiome is inherently complex with high biological diversity, and spatial heterogeneity typically occurring on the submillimetre scale. To study the microbial ecology of soils, and other microbiomes, biomolecules, that is, nucleic acids and proteins, must be efficiently and reliably co‐recovered from the same biological samples. Commercial kits are currently available for the co‐extraction of DNA, RNA and proteins but none has been developed for soil samples. We present a new protocol drawing on existing phenol–chloroform‐based methods for nucleic acids co‐extraction but incorporating targeted precipitation of proteins from the phenol phase. The protocol is cost‐effective and robust, and easily implemented using reagents commonly available in laboratories. The method is estimated to be eight times cheaper than using disparate commercial kits for the isolation of DNA and/or RNA, and proteins, from soil. The method is effective, providing good quality biomolecules from a diverse range of soil types, with clay contents varying from 9.5% to 35.1%, which we successfully used for downstream, high‐throughput gene sequencing and metaproteomics. Additionally, we demonstrate that the protocol can also be easily implemented for biomolecule co‐extraction from other complex microbiome samples, including cattle slurry and microbial communities recovered from anaerobic bioreactors, as well as from Gram‐positive and Gram‐negative pure cultures.     

Electricity-free, sequential nucleic acid and protein isolation

Traditional and emerging pathogens such as Enterohemorrhagic Escherichia coli (EHEC), Yersinia pestis, or prion-based diseases are of significant concern for governments, industries and medical professionals worldwide. For example, EHECs, combined with Shigella, are responsible for the deaths of approximately 325,000 children each year and are particularly prevalent in the developing world where laboratory-based identification, common in the United States, is unavailable (1). The development and distribution of low cost, field-based, point-of-care tools to aid in the rapid identification and/or diagnosis of pathogens or disease markers could dramatically alter disease progression and patient prognosis. We have developed a tool to isolate nucleic acids and proteins from a sample by solid-phase extraction (SPE) without electricity or associated laboratory equipment (2). The isolated macromolecules can be used for diagnosis either in a forward lab or using field-based point-of-care platforms. Importantly, this method provides for the direct comparison of nucleic acid and protein data from an un-split sample, offering a confidence through corroboration of genomic and proteomic analysis. Our isolation tool utilizes the industry standard for solid-phase nucleic acid isolation, the BOOM technology, which isolates nucleic acids from a chaotropic salt solution, usually guanidine isothiocyanate, through binding to silica-based particles or filters (3). CUBRC's proprietary solid-phase extraction chemistry is used to purify protein from chaotropic salt solutions, in this case, from the waste or flow-thru following nucleic acid isolation(4). By packaging well-characterized chemistries into a small, inexpensive and simple platform, we have generated a portable system for nucleic acid and protein extraction that can be performed under a variety of conditions. The isolated nucleic acids are stable and can be transported to a position where power is available for PCR amplification while the protein content can immediately be analyzed by hand held or other immunological-based assays. The rapid identification of disease markers in the field could significantly alter the patient's outcome by directing the proper course of treatment at an earlier stage of disease progression. The tool and method described are suitable for use with virtually any infectious agent and offer the user the redundancy of multi-macromolecule type analyses while simultaneously reducing their logistical burden.     

Method for automated extraction and purification of nucleic acids and its implementation in microfluidic system

A method and a microfluidic device for automated extraction and purification of nucleic acids from biological samples have been developed. The method involves disruption of bacterial cells and/or viral particles by combining enzymatic and chemical lysis procedures followed by solid-phase sorbent extraction and purification of nucleic acids. The procedure is carried out in an automated mode in a microfluidic module isolated from the outside environment, which minimizes contact of the researcher with potentially infectious samples and, consequently, decreases the risk of laboratory-acquired infections. The module includes reservoirs with lyophilized components for lysis and washing buffers; a microcolumn with a solid-phase sorbent; reservoirs containing water, ethanol, and water-ethanol buffer solutions for dissolving freeze-dried buffer components, rinsing the microcolumn, and eluting of nucleic acids; and microchannels and valves needed for directing fluids inside the module. The microfluidic module is placed into the control unit that delivers pressure, heats, mixes reagents, and flows solutions within the microfluidic module. The microfluidic system performs extraction and purification of nucleic acids with high efficiency in 40 min, and nucleic acids extracted can be directly used in PCR reaction and microarray assays.     

NMR studies of dynamics in RNA and DNA by 13C relaxation

RNA and DNA molecules experience motions on a wide range of time scales, ranging from rapid localized motions to much slower collective motions of entire helical domains. The many functions of RNA in biology very often require this molecule to change its conformation in response to biological signals in the form of small molecules, proteins or other nucleic acids, whereas local motions in DNA may facilitate protein recognition and allow enzymes acting on DNA to access functional groups on the bases that would otherwise be buried in Watson-Crick base pairs. Although these statements make a compelling case to study the sequence dependent dynamics in nucleic acids, there are few residue-specific studies of nucleic acid dynamics. Fortunately, NMR studies of dynamics of nucleic acids and nucleic acids-protein complexes are gaining increased attention. The aim of this review is to provide an update of the recent progress in studies of nucleic acid dynamics by NMR based on the application of solution relaxation techniques.     

A comparison of some procedures of salt extraction of nucleic acids from pollen. Elimination of interfering substances from extracts

Some methods were studied which use hot 10% NaCl solution for the extraction of both RNA and DNA from pollen. The raw salt extracts were precipitated with perchloric acid, trichloroacetic acid or ethanol and purified according to the described methods. The nucleic acid hydrolysates were obtained in several ways. In all the samples spectra in the UV-region were measured and the nucleic acid contents were determined according to the absorbance at 260 nm. In order to ascertain the extent of contaminants, the contents of phosphorus, saccharides and proteins were determined. It was found that by the methods studied it is possible to remove some impurities from extracts, but that the extractions of nucleic acids from pollen are not quite quantitative. A part of nucleic acids remained unextracted after the salt extraction in pollen, but it was possible to obtain it only by an additional extraction with 1 N perchloric acid at 75°C.     

Role of carbon nano-materials in the analysis of biological materials by laser desorption/ionization-mass spectrometry

At present, carbon nano-materials are being utilized in various procedures, especially in laser desorption/ionization-mass spectrometry (LDI-MS) for analyzing a range of analytes, which include peptides, proteins, metabolites, and polymers. Matrix-oriented LDI-MS techniques are very well established, with weak organic acids as energy-absorbing substances. Carbon materials, such as nano-tubes and fullerenes are being successfully applied in the small-mass range, where routine matrices have strong background signals. In addition, the role of carbon nano-materials is very well established in the fractionation and purification fields. Modified diamond powder and surfaces are utilized in binding peptides and proteins from complex biological fluids and analyzed by matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS). Polylysine-coated diamond is used for solid-phase extraction to pre-concentrate DNA oligonucleotides. Graphite is useful for desalting, pre-concentration, and as energy-absorbing material (matrix) in desorption/ionization. Carbon nano-tubes in their different derivatized forms are used as matrix materials for the analysis of a range of analytes, such as carbohydrates, amino acids, peptides, proteins, and some environmental samples by LDI-MS. Fullerenes are modified in different ways to bind serum entities analyzed through MALDI/TOF-MS and are subsequently utilized in their identifications. In addition, the fullerenes are a promising matrix in LDI-MS, but improvements are needed.     

Role of carbon nano-materials in the analysis of biological materials by laser desorption/ionization-mass spectrometry

At present, carbon nano-materials are being utilized in various procedures, especially in laser desorption/ionization-mass spectrometry (LDI-MS) for analyzing a range of analytes, which include peptides, proteins, metabolites, and polymers. Matrix-oriented LDI-MS techniques are very well established, with weak organic acids as energy-absorbing substances. Carbon materials, such as nano-tubes and fullerenes are being successfully applied in the small-mass range, where routine matrices have strong background signals. In addition, the role of carbon nano-materials is very well established in the fractionation and purification fields. Modified diamond powder and surfaces are utilized in binding peptides and proteins from complex biological fluids and analyzed by matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS). Polylysine-coated diamond is used for solid-phase extraction to pre-concentrate DNA oligonucleotides. Graphite is useful for desalting, pre-concentration, and as energy-absorbing material (matrix) in desorption/ionization. Carbon nano-tubes in their different derivatized forms are used as matrix materials for the analysis of a range of analytes, such as carbohydrates, amino acids, peptides, proteins, and some environmental samples by LDI-MS. Fullerenes are modified in different ways to bind serum entities analyzed through MALDI/TOF-MS and are subsequently utilized in their identifications. In addition, the fullerenes are a promising matrix in LDI-MS, but improvements are needed.     

DNA extraction from plants: The use of pectinase

Several earlier protocols for extracting plant DNA or RNA do not work well for a variety of plants because contaminating substances coprecipitate with the nucleic acids and, thus, are present even at the last DNA-hydration step. While DNA extraction protocols have been published in which pectinase is employed to break down these contaminating substances, here we present an alternative modified pectinase protocol that potentially uses fewer steps and avoids the use of ethylene glycol monoethyl ether and phenol. DNA analyses results are described for 6 plant species demonstrating that the method works across distantly related plant taxa.     

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.     

Evaluation of six methods for extraction and purification of viral DNA from urine and serum samples

The sensitivity and reliability of PCR for diagnostic and research purposes require efficient unbiased procedures of extraction and purification of nucleic acids. One of the major limitations of PCR-based tests is the inhibition of the amplification process by substances present in clinical samples. This study used specimens spiked with a known amount of plasmid pBKV (ATCC 33-1) to compare six methods for extraction and purification of viral DNA from urine and serum samples based on recovery efficiency in terms of yield of DNA and percentage of plasmid pBKV recovered, purity of extracted DNA, and percentage of inhibition. The most effective extraction methods were the phenol/chloroform technique and the silica gel extraction procedure for urine and serum samples, respectively. Considering DNA purity, the silica gel extraction procedure and the phenol/chloroform method produced the most satisfactory results in urine and serum samples, respectively. The presence of inhibitors was overcome by all DNA extraction techniques in urine samples, as evidenced by semiquantitative PCR amplification. In serum samples, the lysis method and the proteinase K procedure did not completely overcome the presence of inhibitors.     

A streptavidin paramagnetic-particle based competition assay for the evaluation of the optical selectivity of quadruplex nucleic acid fluorescent probes

Although quadruplex nucleic acids are thought to be involved in many biological processes, they are massively overwhelmed by duplex DNA in the cell. Small molecules, able to probe quadruplex nucleic acids with high optical selectivity, could possibly achieve the visualization of these processes. The aim of the method described herein is to evaluate quickly the optical selectivity of quadruplex nucleic acid probes, in isothermal conditions, using widely available materials, small quantities of oligonucleotides and virtually any kind and quantity of biological competitor. The assay relies on the use of streptavidin-coated paramagnetic particles and biotinylated quadruplex forming oligonucleotides, allowing a quick and easy separation of the quadruplex target from the competitor. In the present study, two quadruplex nucleic acids (the DNA and RNA human telomeric repeats) have been used as targets while a duplex DNA oligonucleotide, total DNA, total RNA, another quadruplex nucleic acid and a protein have been used as competitors. The optical selectivity of various probes, displaying different photophysical properties and binding selectivities, has been successfully examined, allowing the identification of a best candidate for further cell microscopy experiments. This assay allows a quick and reliable assessment of the labeling properties of a quadruplex binder in cellular environment conditions. It is an interesting alternative to gel electrophoresis experiments since it is performed in solution, has a well-resolved separation system and allows easy quantifications.     

Problems associated with the labelling of RNA with radioactive precursors in vivo (author's transl)]

The radioactivity of RNA, DNA and proteins in the liver, muscles and cerebrum of 30-day-old rats after labelling with [3H]uridine, [14C]uridine, [3H]cytidine or [3H]orotic acid was measured. It was found that after administration of [3H]uridine, the proteins were 5 - 10 times more radioactive than the RNA. After administration of [14C]uridine, the proteins were 1 - 2 times more heavily labelled than the RNA. Hydrolysis of the proteins followed by chromatography of the amino acids revealed that the protein labelling was mostly due to [3H]glutamate. In the liver, [3H]orotic acid produced very specific labelling of the RNA. The radioactivity of the proteins is very slight. However, the specific labelling of the RNA in the muscles and cerebrum is not so pronounced with this precursor. [3H]Cytidine is an ideal precursor for RNA. The labelling of protein in all three organs examined is very slight, and furthermore, the specific activity of the RNA is 10 - 20 times higher than after labelling with uridine. We were also able to show that after labelling with radioactive uridine, the method of isolation of RNA by alkaline hydrolysis gives incorrect results, because [3H]amino acids interfere with the measurement of the specific activity of the RNA. The heavy labelling of proteins by [3H]-uridine must also be taken into account in histoautoradiography, because our experiments showed that in liver, the proteins in the cell nucleus are 3 times as radioactive as the nucleic acids. The particulate components of the cytoplasm are even 20 times more radioactive than the nucleic acids.     

Extraction and purification of lipoteichoic acids from gram-positive bacteria

Hot and cold, 80% aqueous phenol extraction procedures together with an aqueous extraction technique have been evaluated for the isolation of lipoteichoic acids from the cytoplasmic membrane of Gram-positive bacteria. Lipoteichoic acids of Staphylococcus aureusH, Micrococcus 2102, Bacillus subtilis 168, and Bacillus subtilis W-23 were examined as each of them emphasises a different problem of contamination. The purity of the lipoteichoic acids with respect to cell-wall material, nucleic acid, and protein is discussed together with the criteria of purity which enables critical structural analysis of lipoteichoic acids to be carried out.