Isolation Of Deoxyribonucleic Acid Research Articles

Comparison of undefined medium and its dialyzable fraction for growth of Mycoplasma.

In examining the medium used in cultivation of Mycoplasma for deoxyribonucleic acid isolation, it was found that an aggregate was present which sedimented with the organisms and which was ethyl alcohol-precipitated during deoxyribonucleic acid purification. To eliminate the contaminating material, a method was devised to obtain only the dialyzable constituents of the medium. This report describes the preparation of a dialysate of soy peptone-yeast extract. The medium, obtained by immersion of the encased dehydrated ingredients in sodium chloride solution for 5.5 hr at approximately 80 C, has been employed as the basal medium for cultivation of a number of Mycoplasma species. Comparative growth curves of two saprophytic strains and two parasitic species indicated that multiplication in dialysate, with suitable supplement, followed the pattern typical of the common eubacteria. Thus, by elimination of the sediment which occurred in nondialyzed medium, Mycoplasma could be concentrated without concomitant accumulation of contaminating macromolecules.

Isolation of deoxyribonucleic acid from the yolk platelets of Xenopus laevis oöcyte.

Isolation of deoxyribonucleic acid from the yolk platelets of Xenopus laevis oöcyte.

Isolation of deoxyribonucleic acid and ribosomal ribonucleic acid from rat liver

Isolation of deoxyribonucleic acid and ribosomal ribonucleic acid from rat liver

Production of Escherichia coli as a source of nucleic acids

AbstractPresent‐day chemical and physical studies of the nucleic acid system of bacteria frequently call for quantities of cells which are beyond the capacity of traditional culture methods such as that of the shake flask. The case is therefore argued for an increased use of stirred deep cultures because a greater rate of output, at a higher cell concentration and with improved control of cultural conditions can be achieved. For the isolation of deoxyribonucleic acid and transfer ribonucleic acid, low growth‐rate cells should be the most prolific source: whereas for ribosomes, ribosomal ribonucleic acid and messenger ribonucleic acid, high growth cells should give bigger yields. It is concluded that the information is somewhat obscure on what are the best growth conditions for the production of the related‐enzymes.A batch method for producing low growth‐rate cells of Escherichia coli, in lots of 1 kg dry wt., is then described, followed by a continuous method for producing high growth‐rate cells, with an output of 3 kg of dry cells per day. Both cultures have been designed to give maximum yield of cells by promoting maximum assimilation of the carbon substrate. From the point of view of designing a culture, the section dealing with the continuous process discusses the rationale of medium formulation, the calculation of aeration requirements and briefly reviews earlier work on the kinetics of continuous culture.

Isolation of deoxyribonucleic acid from mycobacteria.

Isolation of deoxyribonucleic acid from mycobacteria.

The isolation, analysis and chemical reactions of deoxyribonucleic acid (DNA)—I.: The isolation and purification of DNA

The methods available for the isolation of DNA from various sources and in amounts varying from 1 kg to single intact molecules are reviewed. Emphasis has been placed on those methods which have been reported since 1960. The methods available for the separation of DNA from ribonucleic acids, polysaccharides, proteins and other cellular material are discussed, together with some of the methods which have been proposed for separating native and denatured DNA.

Isolation of deoxyribonucleic acid and ribosomal ribonucleic acid from bacteria

1. Nucleic acids were released from Escherichia coli by lysing with tri-iso-propylnaphthalene sulphonate and 4-aminosalicylate and then extracting with a phenol-cresol mixture. 2. Nucleic acids were similarly released from Bacillus subtilis after initial treatment with lysozyme. 3. DNA was sedimented after careful precipitation with m-cresol or 2-butoxyethanol (0.1-0.12vol.) in the presence of 20% sodium benzoate. 4. Contaminating ribosomal RNA was removed by precipitation in the presence of 4m-sodium chloride or by extracting DNA with an acetate-butyrate mixture, in which RNA is insoluble. 5. The DNA from B. subtilis has a transforming ability of 0.3-0.6% for the tryptophan marker. 6. Ribosomal RNA was then precipitated with rapidly labelled RNA by the addition of an equal volume of 2-butoxyethanol. 7. There was good separation of the nucleic acids from protein and polysaccharides.

Isolation of deoxyribonucleic acid from mammalian tissues.

1. DNA has been isolated from different mammalian tissues. The DNA preparations were free from RNA, protein and polysaccharides and have a similar range of sedimentation coefficients (approx. 24s). 2. Protein was removed by a two-stage extraction with a phenol-cresol mixture by using a detergent with 4-aminosalicylate in the first stage and sodium chloride in the second. 3. Polysaccharides remained in solution when DNA was precipitated with 2-butoxyethanol in the presence of 0.5m-sodium chloride and 1.5m-sodium benzoate. 4. Ribosomal RNA was removed by precipitation in the presence of 3m-sodium chloride at 0 degrees , when DNA remained soluble.

Use of alkali in a rapid procedure for the isolation of partly-degraded deoxyribonucleic acid: investigation of the method introduced by W. Jones.

AbstractAn investigation has been carried out in order to avoid deamination of cytosine residues in the isolation of deoxyribonucleic acid (DNA) using hot alkali. DNA obtained by the method of W. Jones1 contains much smaller amounts of uracil residues than that generally available commercially. Attempts to modify the method of Jones to yield a completely undeaminated product and at the same time to maintain the simplicity of this process, were not successful; the products were either deaminated to the same extent or contained much protein. It was found that, during the action of alkali at 100° on highly‐polymerised DNA, cytosine residues were deaminated to uracil residues and free adenine and traces of guanine were liberated.

A new method for the isolation of deoxyribonucleic acid from microorganisms

1. An isolation procedure for DNA involving multistep liquid-liquid extraction with an aqueous polymer two-phase system has been developed. By changing the ionic composition of the system the nucleic acids are selectively extracted. 2. The main advantages of the method are that the DNA is not precipitated, no treatment with ribonuclease is needed to remove the bulk of the RNA, and extensive use of organic solvents is avoided. 3. A simple way of removing the phase polymers is presented. 4. The final product has a sedimentation coefficient, s 20,w = 29 S . Protein content is less than 1 % of the DNA. RNA content is less than 5 %. The product has good transforming activity as tested with Bacillus subtilis.

Comparison of physicochemical properties of mitochondrial and nuclear deoxyribonucleic acid from rat liver cells.

THE discovery of deoxyribonucleic acid (DNA) in mitochondria1–9 has opened the way for investigations of the long-standing problem of extra-genome cytogenetic determinants. DNA fibres have been found in mitochondria from tissues belonging to various different classes of animals1. The principal difficulty to be dealt with in the isolation of mitochondrial DNA is caused by the necessity of separating the soluble nuclear DNA, adsorbed on the surface of mitochondria. For this purpose we used a method of purification of mitochondria in ‘Urographin’ density gradient8 or treatment of the mitochondrial fraction with DNase (150 µg/ml. for 1.5 h at 0° C). Fibres of high-polymer deproteinized DNA were obtained by means of the phenol method after Kirby10 and Marmur11. The average DNA content in liver mitochondria amounted to 0.65 µg/mg protein.

Role of enzymes in the isolation of deoxyribonucleic acid with sodium dodecyl sulfate

The role of enzymes in causing the release of DNA from residual protein of the nucleus during the isolation of DNA with sodium dodecyl sulfate has been investigated. It has been concluded from several lines of evidence that the chief factor responsible for the DNA release is catheptic action which occurs in spite of the presence of the dodecyl sulfate. It is also concluded that in the cases of tissues where the release of DNA from residual protein is unsatisfactory, external protease must be added if the isolation is to be satisfactory. If clean DNA is to be isolated from calf thymus, rat liver, or chicken erythrocytes, the gels that are formed on the addition of dodecyl sulfate must be broken, but breaking the gels is not sufficient to insure good removal of residual protein. The mechanism of action of SH compounds in causing degelation of DNA-nucleoprotein gels in sodium dodecyl sulfate has been studied. The total yield of DNA with the sodium dodecyl sulfate method has been studied in the case of calf thymus after the addition of purified DNA to the system, and it has been concluded that (a) the added DNA after heating can become complexed with the DNA present in the tissues if it is homologous, and (b) that on heating the system to the boiling point during the isolation procedure, a firm complex is formed between the histone and the residual protein to which the inherent DNA is already complexed. This results in the loss of most of the DNA owing to precipitation along with the protein during the deproteinization step.

Isolation of deoxyribonucleic acid from mitochondria of chick embryo heart and liver.

Isolation of deoxyribonucleic acid from mitochondria of chick embryo heart and liver.

Isolation of deoxyribonucleic acid and ribosomal ribonucleic acid from Escherichia coli.

Isolation of deoxyribonucleic acid and ribosomal ribonucleic acid from Escherichia coli.

Direct Isolation of Deoxyribonucleic Acid from Cells

THERE are now available several variations of a few reliable methods for the isolation of DNA from tissues. Generally they are based on the preliminary saline extraction of the tissue, in a method first used by Mirsky and Pollister1. The DNA of the nucleoprotein is isolated with one of a number of different reagents, resulting in a preparation of DNA which is considered by all present criteria to be similar to the native material of the cell. Among the many reagents which have been used for the deproteinization of the nucleoprotein have been octyl alcohol–chloroform mixtures2, the detergent sodium dodecyl sulphate3,4 and more recently phenol and p-amino salicylate5, and phenolphthalein diphosphate6. All these methods have been of considerable use in providing DNA which has been shown by various tests to retain biological activity. However, most of the present methods for obtaining DNA from mammalian cells suffer from the same disadvantage. They all permit possible enzyme degradation during the preliminary washing and fractionation of the tissue prior to the extraction of the nucleic acids.

The isolation of deoxyribonucleic acid from plant tissues

A method is described for isolating DNA from tobacco leaves, based on phenol treatment of a leaf homogenate in the presence of sodium diethyldithiocarbamate and phenolphthalein diphosphate. The product is of high molecular weight, free from RNA, and is obtained in high yield. The method has been successfully applied to a number of other plant tissues. Equilibrium density gradient centrifugation of DNA prepared from healthy and tobacco mosaic virus-infected tobacco leaves showed only one class of DNA to be present; hence (a) if chloroplasts as well as nuclei contain DNA, the base compositions are closely similar, and (b) there is no evidence for production of a specific DNA as a consequence of virus infection.

The deoxyribonucleic acids of some protozoa and a amould.

SYNOPSIS. Methods have been developed for the isolation of deoxyribonucleic acids (DNA) from Tetrahymena pyriformis, Polytomella papillata, and Aspergillus tamarii. The DNA from these organisms contained adenine (A), thymine (T), guanine (G) and cytosine (C), and the usual base‐pairing relationship, i.e., A = T; G = C was found. The ratio A + T/ G + C was 2.42, 1.42, and 1.08 for the DNA of the three organisms respectively. 5‐Methylcytosine was absent in all cases.

A COMPARISON OF METHODS FOR THE ISOLATION OF DEOXYRIBONUCLEIC ACID FROM SMALL AMOUNTS OF RAT TISSUE

Different procedures were studied for the isolation of deoxyribonucleic acid from liver and intestinal mucosa of the rat. The DNA preparations were compared with respect to intrinsic viscosities, extinction coefficients, nitrogen and phosphorus content, and base ratios. Hydrolyzates of the DNA samples contained amino acids indicating the presence of protein or peptides in the preparations. An improved technique was developed for the elution of purines and pyrimidines from paper chromatograms.

A COMPARISON OF METHODS FOR THE ISOLATION OF DEOXYRIBONUCLEIC ACID FROM SMALL AMOUNTS OF RAT TISSUE

Different procedures were studied for the isolation of deoxyribonucleic acid from liver and intestinal mucosa of the rat. The DNA preparations were compared with respect to intrinsic viscosities, extinction coefficients, nitrogen and phosphorus content, and base ratios. Hydrolyzates of the DNA samples contained amino acids indicating the presence of protein or peptides in the preparations. An improved technique was developed for the elution of purines and pyrimidines from paper chromatograms.

Observations on the use of phenol for the isolation of deoxyribonucleic acid

A simple, rapid method for the isolation of DNA from mammalian cells and from pneumococci is described. It involves repeated extraction of the cells, suspended in buffered 1 M NaCl, with water-saturated phenol. The product obtained is remarkably free of contamination by polypeptides, polysaccharides and RNA. The sedimentation characteristics of DNA isolated by this procedure have been investigated in the analytical ultracentrifuge, and the possible significance of the results is discussed.