Nucleic acid cross-linking methods

Another coupling method, i.e. cross-linking or entrapment in polymeric films, which has been used to create a more permanent nucleic acid surface, is described in some chapters (e.g. conductive electroactive polymers for DNA immobilization and self-assembly DNA-conjugated polymers). One chapter reviews the basic characteristics of the biotin-(strept)avidin system laying the emphasis on nucleic acids apphcations. The biotin-(strept)avidin system can be also used for rapid prototyping to test a large number of protocols and... 

The unique properties of oligonucleotides create cross-linking options that are far different from those of any other biological molecule. Nucleic acids are the only major class of macromolecule that can be specifically synthesized in vitro by enzymatic means. The addition of modified nucleoside triphosphates to an existing DNA strand by the action of polymerases or transferases allows addition of spacer arms or detection components at random or discrete sites along the chain. Alternatively, chemical methods that modify nucleotides at selected functional groups can be used to produce spacer arm derivatives or activated intermediates for subsequent coupling to other molecules. 

Oligonucleotides containing 4-thiothymidine and 4-thiouridine have been previously used for LiV cross-linking studies with a view to the study of nucleic acid structures and DNA-protein interactions. A recent publication describes the synthesis of a. set of deoxyuridine derivatives (174, 175, 176) containing C5-pendant thiothymine residues suitable for incorporation into oligonucleotides using chemical methods. 

Stable joining of protein to nucleic acid can be demonstrated by several procedures. Perhaps the easiest approach is a simple phenol extraction method that gives accurate and reproducible results. This method is based on the observation that proteins and nucleic acids ordinarily can be separated by phenol extraction protein migrates into the phenol layer whereas nucleic acids remain in the aqueous phase. However, upon linkage of a tRNA to a synthetase the nucleic acid migrates with the protein into the phenol layer. A similar observation was made earlier by Markovitz, who showed that DNA cross-linked to DNA polymerase is also extracted into phenol. ... 

Corticosteroid receptor, 76 Corticosterone, 76 Creatine kinase, 21 Cross linking, 90, 641, 642 antibody, 501-504 artifacts, 174, 175 characterization, 172-180 efficiency, 170-172 intermolecular, 90 irradiation, 170-172 methods for, 170-172 nucleic acid-protein, 168-180 of oligonucleotides, 676 quantitation, 171, 178 regions, 175, 176 procedures, 176-178 Curtius-Schmidt rearrangement, 78 w-Cyanobenzyltriphenylphosphonium bromide, 125, 127 Cyanogen bromide, 114 tn-Cyanophenol, 122-124 m-(3-Cyanophenoxy)propylamine hydrobromide, 124... 

Electrophoresis A family of separation methods based on the motion induced in particles by an applied, uniform electric field. The most common application of electrophoresis is gel electrophoresis, in which sample (typically proteins, amino acids, nucleic acids, etc.) is applied to a channel that is formed in a cross-linked polymer, usually polyacrylamide or agarose (the gel). The speed at which the individual species move through the gel under the influence of the field is determined largely by the size of the species, as expressed by the mass-to-charge ratio. After separation, the individual species usually appear as discrete bands that may be better visualized by staining with ethidium bromide, silver, or Coomassie Brilliant Blue dye. Other related... 

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