Nucleic acid preparation

Breter, H.-J., Seibert, G., and Zahn, R. K., Single-step separation of major and rare ribonucleosides and deoxyribonucleosides by high-performance liquid cation-exchange chromatography for the determination of the purity of nucleic acid preparations, ]. Chromatogr., 140, 251, 1977. 

Values found for the molecular weight of deoxyribonucleic acids also vary considerably, but probably lie between 1.0 X 106 and 4.4 X 106. Various difficulties encountered in making such measurements have been discussed by Jordan,244 and it is probable that more reliable information will be obtained only when the behavior of polyelectrolytes in general is better understood. Certain of the techniques used are useful in detecting differences between different nucleic-acid preparations, but the discrepancies between the values given by different methods of measurement appear to vary with the degree of polymerization.246... 

Figure 6. Affinity chromatography of EGD from Clostridium thermocellum. Nucleic acid preparation, heat treatment and ammonium sulfate precipitation (0-70%, 70-100%) were carried out as described (10). The final precipitate ( 50 mg protein), dissolved in 50 mM sodium acetate, pH 5.0, was applied (after centrifugation) on the affinity column (2 x 25 cm) (4 -aminobenzyl l-thio-/ -cellobioside coupled to Sepharose 4B) (11). Protein was monitored at 280 nm and the activity of the fractions (2 ml) determined using 2 -chloro-4 -nitrophenyl / -cellobioside (pH 6.5, 25°C) as described in the text. Elution with 10 mM G2 was started as indicated.

Write all observations of your nucleic acid preparation in your notebook. Prepare a flowchart for the isolation procedure. Explain the purpose of each step and the role played by each reagent. Speculate on any damage that may have altered the molecular structure of the DNA. 

B 2. How could RNA be removed from the nucleic acid preparation ... 

A better method for highly parallel genetic analysis is needed. One with single molecule sensitivity that eliminates both the requirement for target amplification and the need for a target to be chemically modified or labeled for its detection would be ideal. Innovations in array construction, sample nucleic acid preparation... 

The discovery of a small proportion of a nucleoside containing thymine42 in the ribonucleic acid of two strains of Escherichia coli, in Aerobacter aero-genes, and in commercial, yeast-ribonucleic acid emphasizes the point made previously,26-28 namely, that the nucleic acids may contain constituents other than those heretofore identified. Alkaline hydrolysis of the ribonucleic acid from E. coli gave nucleotides42 (probably the 2- and 3-phosphate esters) which were converted to the nucleoside with prostatic phospho-monoesterase.62 Enzymic hydrolysis of the nucleic acid preparation also led to the nucleoside, which was degraded further to thymine by hydrolysis with perchloric acid.42 There can be little doubt that this carbohydrate derivative of thymine is intimately bound as part of the polynucleotide chain of this particular ribonucleic acid. 

Walsh (2003) defined biopharmaceuticals as therapeutic protein or nucleic acid preparations made by techniques involving recombinant deoxyribonucleic acid (DNA) technology. Therapeutic proteins include blood clotting factors and plasminogen activators, hemopoietic factors, hormones, interferons and interleukins, and monoclonal antibodies (LeVine, 2006). Over time, the term biopharmaceutical has broadened, and, in addition to proteins and nucleic acids, now includes bacteriophages, viral and bacterial vaccines, vectors for gene therapy, and cells for cell therapy (Primrose and Twyman, 2004). Attention here focuses on proteins, since the majority of approved biopharmaceuticals are proteins. 

II. Lysis Enriched Cytoplasmic Nucleic Acid Preparation... 

For the estimation of traces of proteins in nucleic acid preparations, a pulse polarographic method is more suitable than classical polarography... 

It enables the detection of proteins in concentrations of approximately 0.1 fxglml, and a few micrograms of the sample suffice for the estimation of 1% protein in a nucleic acid preparation. 

Photohaptens photobiotin or photodigoxigenin can be introduced by simple methods photohaptens react with many different molecules and nucleic acid preparation should be very pure detectability somewhat inferior... 

Once the bacterial cells have been lysed and a crude preparation of plasmid has been obtained, several other methods are available for purification of plasmid from the crude nucleic acid preparation, such as cesium chloride ultracentrifugation, gel filtration/size exclusion chromatography, anion exchange (used in the Qiagen kit mentioned above), and HPLC (Table 2) (see Note 2). 

Gel filtration or size exclusion isolation of plasmid from the crude nucleic acid preparation offers the advantages of good separation of plasmid from genomic DNA and RNA and a simple, inexpensive procedure (165,166). The use of Sephacryl S-1000 Superfine matrix allows excellent separation of plasmid from RNA. When this separation is optimal, the use of RNase can be omitted from the isolation procedure, which is important for clinical considerations. This... 

In many cases absolute purity of DNA (or RNA) is not necessary for use as templates in PCR reactions. However, if impure templates are used, care must be taken to remove inhibitors of Taq polymerase in the nucleic acid preparation. For further discussion of inhibitory contaminants and easier protocols designed for PCR work, see sample preparation methods (15,18,19) and Chapters 2 and 3 of this volume. 

The nucleic acids were discovered by Miescher in 1868-1869, when he isolated from pus cell nuclei a material which contained phosphorus, was soluble in alkali, but precipitated under acidic conditions. This material was subsequently prepared from other sources and when freed from protein it was called nucleic acid, a term introduced by Altman in 1889. The classical preparations of nucleic acid from yeast yielded a product which we now recognize as ribonucleic acid (RNA). The nucleic acid prepared from thymus glands, thymonucleic acid, was also extensively studied this material [which, in present terms, was deoxyribonucleic acid (DNA)) was different from yeast nucleic acid. From hydrolysates of these preparations the heterocyclic bases were isolated and characterized. At one time, yeast and thymus nucleic acids were thought to be representative of plant and animal nucleic acids, respectively (3). By 1909, it was apparent that yeast nucleic acid contained adenine, guanine, cytosine, uracil, phosphoric acid, and a sugar which Levene showed at that time to be D-ribose. Thymonucleic acid yielded adenine, guanine, cytosine, thymine, phosphoric acid, and a sugar which was not identified correctly until 1929, when it was characterized as 2-deoxy-D-ribose. 

The biuret reaction [alkaline Cu(II)] and the arginine test [102] are used to detect peptides in nucleic acid preparations. Two other colour reactions permit either type of nucleic acid to be detected in the presence of the other one is due to Dische and the other is the phloroglucinol test of v. Etjler and Hahn. 

Ito, Y. and C.A. Evans. 1961. Induction of tumors in domestic rabbits with nucleic acid preparations from partially purified Shope papilloma virus and from extracts of the papilloma of domestic and cottontail rabbits. J. Exper. Med. 114 485-500. 

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