Nucleic acid denaturation

The Helix-to-Random-Coil Transition Nucleic Acid Denaturation... 

Nucleic acid denaturation , Comments Solid State Phvs.. 2, 49... 

With the advent of nucleic acid hybridization as a pivotal technique in recombinant DNA technology, the theoretical and practical aspects of nucleic acid denaturation—renaturation have received renewed, more rigorous attention (11). How does a smaU piece of DNA (or RNA) find its complementary sequence from among often astronomical numbers of similar sequences Finding a 1000 base stretch in the human genome would be like finding a needle in a 2-ton haystack. 

Fig. 1. Southern blot analysis of DNA showing (a) step 1, an agarose gel containing separated restriction fragments of DNA, denoted by (—), which is immersed in NaOH to denature the double-stranded stmcture of DNA, and then transferred by capillary flow to a nitrocellulose filter. In step 2, the bound DNA is allowed to hybridize to a labeled nucleic acid probe, and the unbound probe is washed off In step 3, the filter is placed into contact with x-ray film resulting in (b) bands of exposure on the film which are detected after development and correspond to regions where the restriction fragment is...

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... 

The most important product of the hexose monophosphate pathway is reduced nicotinamide-adenine dinucleotide phosphate (NADPH). Another important function of this pathway is to provide ribose for nucleic acid synthesis. In the red blood cell, NADPH is a major reducing agent and serves as a cofactor in the reduction of oxidized glutathione, thereby protecting the cell against oxidative attack. In the syndromes associated with dysfunction of the hexose monophosphate pathway and glutathione metabolism and synthesis, oxidative denaturation of hemoglobin is the major contributor to the hemolytic process. 

Abrams, E.S. and Stanton, V.P. Jr (1992) Use of denaturing gradient gel electrophoresis to study conformational transitions in nucleic acids. Methods inEnzymology 212, 71-104. 

Myers, R.M., Fischer, S.G., Lerman, L.S. and Maniatis, T. (1985a) Nearly all base substitutions in DNA fragments joined to a GC-clamp can be detected by denaturing gradient gel electrophoresis. Nucleic Acids Research 13, 3131-3145. 

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. 

In the past, dissociation of the nucleoprotein complex has been brought about by salt solutions or by heat denaturation,129 but, more recently, decomposition has been effected by hydrolysis with trypsin,126 or by the use of dodecyl sodium sulfate130 or strontium nitrate.131 Some virus nucleoproteins are decomposed by ethyl alcohol.132 This effect may be similar to that of alcohol on the ribonucleoproteins of mammalian tissues. If minced liver is denatured with alcohol, and the dried tissue powder is extracted with 10% sodium chloride, the ribonucleoproteins are decomposed to give a soluble sodium ribonucleate while the deoxyribonucleoproteins are unaffected.133 On the other hand, extraction with 10 % sodium chloride is not satisfactory unless the proteins have first been denatured with alcohol. Denaturation also serves to inactivate enzymes of the tissues which might otherwise bring about degradation of the nucleic acid during extraction. 

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