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.     

Comparison of nucleic acid hybridisation and other tests for detecting tobacco rattle virus in narcissus plants and potato tubers

All isolates of tobacco rattle virus (TRV) found in naturally infected narcissus leaves produced nucleoprotein particles, mostly in large concentrations but, because of antigenic diversity, less than half of the isolates were identified by immunosorbent electron microscopy (ISEM) and still fewer by enzyme-linked immunosorbent assay. All were identified by a nucleic acid hybridisation test in which DNA complementary to RNA-1 of strain PRN of TRV was allowed to react with nucleic acid extracted from leaf tissue. Spraing-affected tubers in some potato stocks yielded only NM isolates of TRV. These isolates do not produce virus particles and they were therefore not detected by ISEM. The infectivity of nucleic acid extracts from recently harvested tubers with spraing symptoms was much greater than that of extracts prepared from tubers after 8 months' storage. In other potato stocks, some spraing-affected tubers contained NM isolates and the rest contained particle-producing isolates (M isolates) of TRV. The infectivity of sap and of nucleic acid, extracted 7 months after harvest from tubers infected with M isolates, was much greater than that of nucleic acid extracted from comparable tubers infected with NM isolates. TRV was detected by nucleic acid hybridisation in extracts of almost all tubers containing either M or NM isolates, even when the tubers were not tested until 7–8 months after harvest. The probable sequence of events occurring after tubers are infected with TRV is outlined, and it is suggested that the virus will rarely become established in fields as a result of planting infected tubers.     

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.     

The effects of kinetin and contaminating bacteria on the incorporation of 32P-orthophosphate into various fractions of nucleic acid extracted from radish leaves

Summary By comparing ³²P-orthophosphate incorporation into nucleic acid extracts of sterile and non-sterile radish leaf discs, it was shown that contaminating bacteria cause a 2–4 fold increase in the rate of precursor incorporation and alter the pattern of label distribution after fractionation of the extracts by sedimentation through sucrose gradients or chromatography on MAK¹ columns. Using sterile senescing radish leaf discs, a stimulation of ³²P-orthophosphate incorporation into various fractions of nucleic acid was observed as a result of kinetin treatment.     

High molecular weight phosphorus compound in nucleic acid extracts of the slime mold Physarum polycephalum

Orthophosphate labeled with (32)P was added to the growth medium of the plasmodia of Physarum polycephalum. The nucleic acid extracts of such plasmodia contained (32)P that was not removed by nuclease, protease, or amylase. This labeled material was shown to be separable from nucleic acids, could be eluted from a methylated albumin-kieselguhr column at 0.5 m NaCl, was of high molecular weight, and had several characteristics in common with polyphosphate. A fraction of this polyphosphate-like material was also found in extracts of isolated nuclei.     

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.     

Two-dimensional gel electrophoresis for identifying proteins that bind DNA or RNA

Electrophoretic mobility shift assays (EMSAs) are commonly used to analyze nucleic acid-protein interactions. When nucleic acid is bound by protein, its mobility during gel electrophoresis is reduced. Similarly, the final position of protein within a complex is shifted when compared to its free state. Here we provide a protocol for a simple approach that uses these mobility differences to identify nucleic acid-binding proteins. Following EMSA, denaturing gel electrophoresis is implemented to provide a second dimension of separation. Protein that binds a specific nucleic acid is identified as a spot(s) whose presence at a particular position(s) is dependent on nucleic acid within the initial binding reaction. The polypeptide in a spot can be subsequently identified by mass spectrometry. As EMSAs can be performed using partially purified or cell extracts, this approach substantially reduces the need for protein purification. It should facilitate the identification of a nucleic acid-binding protein within approximately 4 d.     

Cas12a-based on-site,rapid detection of genetically modified crops

A CRISPR/LbCas12a-based nucleic acid detection method that uses crude leaf extracts as samples and is rapid (≤40 min for a full run) and highly sensitive (0.01%) can be used to monitor genetically modified organisms in the field.     

Further investigation of the biosynthesis of caffeine in tea plants (Camellia sinensis L.). Methylation of transfer ribonucleic acid by tea leaf extracts.

1. The tRNA methyltransferase activity in vitro of leaves, cotyledons and roots of 85-day-old tea seedlings was studied. 2. The activity of extracts prepared from tea leaves with Polycar AT (insoluble polyvinylpyrrolidine) had optimum pH7.7 and was greatly influenced by thiol compounds, but only slightly by metal ions and ammonium acetate. 3. The activities of extracts, expressed per mg of protein, were as follows: roots greater than leaves greater than cotyledons. The only methylated base isolated after incubation with these preparations was 1-methyladenine. 4. The results did not support the view of involvement of methylation of nucleic acids in caffeine biosynthesis in tea plants. In contrast, it is suggested that theophylline is synthesized from the specific methylated precursor in nucleic acids, namely 1-methyladenylic acid, via 1-methylxanthine.     

A cellular gene encodes scrapie PrP 27-30 protein

A clone encoding PrP 27-30, the major protein in purified preparations of scrapie agent, was selected from a scrapie-infected hamster brain cDNA library by oligonucleotide probes corresponding to the N terminus of the protein. Southern blotting with PrP cDNA revealed a single gene with the same restriction patterns in normal and scrapie-infected brain DNA. A single PrP-related gene was also detected in murine and human DNA. PrP-related mRNA was found at similar levels in normal and scrapie-infected hamster brain, as well as in many other normal tissues. Using antisera against PrP 27-30, a PrP-related protein was detected in crude extracts of infected brain and to a lesser extent in extracts of normal brain. Proteinase K digestion yielded PrP 27-30 in infected brain extract, but completely degraded the PrP-related protein in normal brain extract. No PrP-related nucleic acids were found in purified preparations of scrapie prions, indicating that PrP 27-30 is not encoded by a nucleic acid carried within the infectious particles.     

Preparation and properties of a poly A resin and its use in the isolation of naturally occurring poly U sequences

A method for the covalent attachment of poly A, as well as other nucleic acids and nucleosides, to a methylene dianiline derivative of starch is described. The properties of this poly A resin and its use for the recovery of poly U sequences from both nuclear and cytoplasmic extracts of HeLa cells is described.     

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.     

The removal of nucleic acids from microbial extracts by precipitation with lysozyme

It is usually necessary to remove nucleic acids from microbial extracts in order to avoid their interference with the isolation of enzymes from the extract. This may be particularly important where the purification procedure includes chromatography on an anion-exchange column such as DEAE-Sephadex (1). Methods that have been used have included precipitation with Mn, destruction of the nucleic acids with nucleases, and precipitation with basic substances, usually protamine sulfate or streptomycin sulfate. It is likely that there may be advantages, in some cases at least, in using other basic proteins for this purpose, since the results obtained with the previous methods have not always been satisfactory. The procedure described in this paper utilizes high concentrations of lysozyme to precipitate nucleic acids from a bacterial extract. The separation obtained with lysozyme was efficient, reproducible, and superior to the separation obtained with protamine sulfate.     

Hypothalamo-hypophysial-thymic axis in the rat]

Thymic extracts, with proved immunologic properties, decrease, in vitro, RNA and DNA synthesis of hypothalamic fragments of adult rat and have no effect on hypophysis culture; we found a similar action in vivo. Growth hormone inhibits the nucleic acids synthesis of thymic fragments; no action is found with hypothalamic extracts. Hypothesis, that thymic activity is dependent on hypophysis with retroaction on hypothalamus, is discussed.     

Rapid indexing of the sunblotch disease of avocados using a complementary DNA probe to avocado sunblotch viroid

A routine procedure has been established for the sensitive and specific detection of avocado sunblotch viroid in partially purified nucleic acid extracts of avocado leaves by hybridisation analysis with ³²P-complementary DNA prepared against the purified viroid. Avocado sunblotch viroid was shown to be present in 12 avocado trees that had indexed positive in a biological test for sunblotch disease but was absent from 10 trees that indexed negative. The complete correlation between sunblotch disease and the presence of viroid indicates that the complementary DNA hybridisation assay procedure can be used for the indexing of sunblotch disease. The overall procedure of leaf extraction and hybridisation analysis can be completed in 5 days and is to be compared with up to 2 yr required for indexing by biological methods. The level of avocado sunblotch viroid in partially purified nucleic acid extracts of a number of different sources of sunblotch infected avocado leaves was found to vary 10 000-fold from 0.2% to 2 × 10^-5% by weight. The lower limit of detectability of the viroid by the hybridisation assay is considered to be about 10^-5% by weight; this is at least 10³ times more sensitive than the detection of the viroid by polyacrylamide gel electrophoresis of the leaf nucleic acid extracts followed by staining.     

Isolation of nucleic acids from plants by differential solvent precipitation.

The purification of nucleic acids from plant tissue is often made difficult by the presence of contaminating carbohydrate polymers and polyphenols. A procedure for the simultaneous isolation of DNA and translatable RNA from plants is described. The method removes most of the polysaccharides and polyphenols extracted with nucleic acids in a single step by taking advantage of differences in solubility of these compounds in the solvent 2-butoxyethanol. Stepwise addition of 2-butoxyethanol to phenol extracts of specific ionic strength precipitates nucleic acids largely free of contaminants. Subsequent separation of RNA from DNA by precipitation with LiCl was optimised to give a high recovery of translationally active RNA. Successful isolation of nucleic acids from strawberry (Fragaria X ananassa) receptacle, a particularly recalcitrant tissue, and from a range of tissues of other plant species demonstrates the general applicability of the method.     

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.     

Detection of unusual nucleic acids with monoclonal antibodies specific for triplexes, poly(ADP-ribose), DNA-RNA hybrids, and Z-DNA

Monoclonal antibodies which are specific for several unusual nucleic acids are now available. These include Jel 318 which is specific for triplexes, ADP-1 specific for poly(ADP-ribose), Jel 99 specific for RNA-DNA duplexes, and Jel 150 specific for Z-DNA. With the aid of these antibodies and an immunoblotting procedure, unusual nucleic acids can be detected and the amount estimated from a variety of sources. The method involves binding the nucleic acid to either nitrocellulose or Zeta Probe (a cationic nylon membrane), probing with the appropriate monoclonal antibody, followed by addition of an 125I-labeled anti-mouse second antibody. The blot is then developed by autoradiography. The technique is extremely sensitive and can be used to estimate unusual nucleic acids from crude cell extracts.