Denaturation of nucleic acids

Like proteins, nucleic acids can undergo denatur-ation. The strands of the double helix of DNA are separated and the double-stranded regions of RNA molecules "melt." Denaturation can be accomplished by addition of acids, bases, and alcohols or by removal of stabilizing counter ions such as Mg2+. The product is a random coil and denaturation can be described as a helix —> coil transition. Denaturation of nucleic acids by heat, like that of proteins, is cooperative (Chapter 7, Section A,3) and can be described by a characteristic melting temperature. 

Non-solvent processes have been developed in answer to the high cost of large-scale solvent extraction. Holmes and Lim [61] described the enzymatic process used at Zeneca for the recovery of PHB and PHBV. First, a high-temperature (100 to 150 °C) treatment of the cells provokes cell lysis and denaturation of nucleic acids, which could otherwise interfere with the subsequent steps. Non-PHA biomass is then solubilized with proteolytic enzymes (pepsin, trypsin, papain, others, and mixtures thereof) and anionic surfactants. Concentration of PHA by centrifugation is finally followed by bleaching with H2O2. 

Factors to be considered in maldng the selection of chromatography processing steps are cost, sample volume, protein concentration and sample viscosity, degree of purity of protein product, presence of nucleic acids, pyrogens, and proteolytic enzymes. Ease with which different types of adsorbents can be washed free from adsorbed contaminants and denatured proteins must also be considered. 

Polymers of amino acids, like polymers of nucleic acids, have both ionic and hydrophobic character. Unlike the polymers of nucleic acids, amino acid polymers may carry either a negative or a positive (or zero) net charge, are far more subject to irreversible denaturation and oxidation, and are labile to... 

In general, the mechanism of heat and alkaline solution for DNA extraction may be based upon a hypothesis, previously proposed for the AR technique.32 Strong alkaline solution may denature and hydrolyze proteins, resulting in breaking cell and nuclear membranes as well as disrupting cross-linkages due to formalin fixation. It is no surprise to observe the similarity between retrieval of nucleic acid and retrieval of protein (antigen) based on a similar chemical reaction of formaldehyde with these two kinds of macromolecules (Fig. 3.1).15"19... 

Hybridization the process of a single-stranded nucleic acid molecule binding with a complementary single strand of nucleic acid to form a stable double-stranded molecule. Hybridization is temperature dependent, so DNA s that hybridize strongly at low temperature can be temporarily separated (denatured) by heating. 

The manufacture and processing of the protein microarray should be conducted in such a manner that the arrayed proteins remain in their native and active state. For most proteins, this usually means the hydrated state in order to avoid surface denaturation. For antibody arrays which are perhaps more forgiving than other proteins, it has been our experience that while these could be stored cold and dry, it is most important to rehydrate them prior to use. This process is in sharp contrast to the preparation of nucleic acid arrays in which strand melting or denaturahon is necessary to achieve optimal binding to the solid support. While the hybridization process is well understood and can be controlled under thermodynamic principles, the folding and renaturation of proteins on planar (microarray) surfaces is under study. 

The general procedure is as follows. A sample of nucleic acid, which may contain a particular sequence of interest (the target sequence), is denatured and affixed to a... 

Hybridization measurements have been used in many studies of homology of nucleic acids from different species. A nucleic acid is cut (e.g., by sonic oscillation) into pieces of moderate length ( 1000 nucleotides) and is denatured. The denatured DNA fragments are mixed with denatured DNA of another species. Nucleotide sequences that are closely similar between species tend to hybridize, whereas sequences that are... 

Whereas proteins have their low energy absorption band at 280 nm, polynucleotides typically have maxima at 260 nm (38,500 cm ). A phenomenon of particular importance in the study of nucleic acids is the hypochromic effect. In a denatured polynucleotide the absorption is approximately the sum of that of the individual components. However, when a double helical structure is formed and the bases are stacked together, there is as much as a 34% depression in the absorbance at 260 nm. This provides the basis for optical measurement of DNA melting curves (Fig. 5-45).45,86 The physical basis for the hypochromic effect is found in dipole-dipole interactions between the closely stacked base pairs.7,86,87... 

Because of their relatively low molecular weight (70 to 90 nucleotide residues), transfer ribonucleic acids are of special interest for 13C NMR investigations [769, 778, 782-784] of nucleic acids. Using a tube of 20 mm o.d., a sample of thermally denatured yeast... 

Figure 16.5 (a) The native folded state of the protein and the unfolded, denatured state following the thermally-induced structural change (b) the duplex state of nucleic acids, stable at low temperatures, in which the bases are paired and stacked, and the monomer states following the thermal disruption in which the bases are unpaired and randomly arranged along the backbone. 

In summary the deleterious effects of alkali on the proteins and the large-scale impracticality of the heat-shock (endogeneous ribonuclease) process, plus the accompanying proteolysis and denaturation of proteins, clearly indicate the need for better methods to facilitate the large-scale separation of nucleic acids from yeast proteins. 

This increase in the absorption spectrum following denaturation (destruction of secondary structure) is termed the hyperchromic effect (Fig. V-9). Conversely, the decrease in the absorption spectrum on renaturation of these types of nucleic acids (restoration of secondary structure) is termed the hypochromic effect. These effects are observed in Experiment 19. 

Fig. 2. Effect of native calf thymus DNA, denatured calf thymus DNA and yeast RNA on the visible absorption spectrum of tilorone in 0.01 M Tris-HCl (pH 7.0). Curve 1 is the spectrum of free tilorone (4.25 x 10-4 M). Other curves depict the spectra of tilorone in the presence of yeast RNA (curve 2), denatured DNA (curve 3) and native DNA (curve 4). Molar concentrations of nucleic acids (2 x 10 3 M) refer to phosphorous content of the polymer...

Metal ions are usually required to promote and stabilize functionally active or native conformations of nucleic acids, as well as to mediate nucleic acid-protein interactions. However, metal ions can also cause structural transformation of nncleic acids, or denature their native structures. In addition to structural roles, some metal compounds can indnce cleavage (i.e. scission, fragmentation, or depolymerization) and modification (withont cleavage) of nucleic acids. Metal-nucleic acid interactions can be either nonspecific or dependent on the chemical nature of nucleotide residues, nucleic acid sequence, or secondary and/or tertiary structure of nucleic acids. The specificity of these interactions is dependent... 

Electrophoresis is without doubt the most convenient and accurate method for determining the size of nucleic acids (Sect. 4.2). For double stranded nucleic acids, electrophoresis is carried out under non-denaturing conditions using a polyacrylamide or agarose matrix, depending on the size of the nucleic acid. For single stranded nucleic adds, electrophoresis should be conducted in the presence of urea, to disrupt any partially based-paired structures that may influence mobility. In both cases, the samples of interest should be run together with molar mass standards, which can either be prepared in the laboratory or purchased. 

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