Detection of nucleic acids

The emission yield from the horseradish peroxidase (HRP)-catalyzed luminol oxidations can be kicreased as much as a thousandfold upon addition of substituted phenols, eg, -iodophenol, -phenylphenol, or 6-hydroxybenzothiazole (119). Enhanced chemiluminescence, as this phenomenon is termed, has been the basis for several very sensitive immunometric assays that surpass the sensitivity of radioassay (120) techniques and has also been developed for detection of nucleic acid probes ia dot-slot. Southern, and Northern blot formats (121). 

Figure 1. Dyes used for detection of nucleic acids by fluorescence.

The previous biomarkers relate to phenotypic assessments of microbial diversity and most will probably measure a restricted part of the total microbial pool, since not alt markers will be expressed uniformly by every cell. In contrast, methods involving the detection of nucleic acids may be directly applicable to all microorganisms provided that the complete extraction of DNA (lysis of cells) or permea-bilization of cells can be achieved. 

The analysis of extraterrestrial matter is concentrated on the detection of nucleic acid and protein building blocks, i.e., N-heterocycles and amino acids. The search for such compounds began immediately after the fall of the Murchison meteorite. Twenty-two amino acids were detected in it as early as 1974 eight of them pro-teinogenic, ten which hardly ever occurred in biological material, and four which were unknown in the biosphere. Up to now, about 70 amino acids have been identified (Cronin, 1998), the most common being glycine and a-aminoisobutyric acid. The latter is a branched-chain amino acid with the smallest possible number of carbon atoms. The most frequently found amino acids occur in concentrations of... 

Similar techniques can be used to devise (strept)avidin-biotin assay systems for detection of nucleic acid hybridization. DNA probes labeled with biotin can be detected after they bind... 

Filter membranes HRP/H202/luminol AP/dioxetanes Detection of nucleic acids and proteins... 

Methods currently available for chemiluminescent detection of nucleic acids are not based on derivatization techniques that directly recognize one of the nucleic acid bases or nucleotides. For chemical derivatization-based chemiluminescent detection, the specific reactivity of alkyl glyoxals and arylglyoxals with adenine or guanine nucleotides has been investigated. 

Huhtinen P, Vaamo J, Soukka T et al (2004) Europium(III) nanoparticle-label-based assay for the detection of nucleic acids. Nanotechnology 15 1708-1715... 

Ho HA, Dore K, Boissinot M, Bergeron MG, Tanguay RM, Boudreau D, Leclerc M (2005) Direct molecular detection of nucleic acids by fluorescence signal amplification. J Am Chem Soc 127 12673-12676... 

Dore K, Leclerc M, Boudreau D (2006) Investigation of a fluorescence signal amplification mechanism used for the direct molecular detection of nucleic acids. J Fluoresc 16 259-265... 

Nucleic acid structures and sequences primary and secondary structure of DNA fragments, translocation of genes between two chromosomes, detection of nucleic acid hybridization, formation of hairpin structures (see Box 9.4), interaction with drugs, DNA triple helix, DNA-protein interaction, automated DNA sequencing, etc. 

In situ hybridization may be defined as the detection of nucleic acids in situ in cells, tissues, chromosomes, and isolated cell organelles. The technique was described in 1969 by two separate groups who demonstrated repetitive riboso-mal sequences in nuclei of Xenopus oocytes using radiolabeled probes (1,2). Refinements in recombinant DNA technology and the development of nonisotopic probe labeling and detection (3) obviate the need for radiation protection and disposal facilities, and have converted nonisotopic in situ hybridization (NISH) from a purely research technique to one that can be used in routine laboratory testing. 

Similar techniques can be used to devise avidin—biotin assay systems for detection of nucleic acid hybridization. DNA probes labeled with biotin can be detected after they bind their complementary DNA target through the use of avidin-labeled complexes (Bugawanefrz/., 1990 Lloyd etal., 1990). Direct detection of hybridized probes can be accomplished, in a manner similar to that for LAB, by incubating with an avidin-enzyme conjugate followed by substrate development. BRAB-like and ABC-like assays also can be utilized to further enhance a DNA probe signal (Chapter 17, Section 2.3). 

Holtke, H.-J., Seibl, R., Burg, J., Miihlegger, K., and Kessler, C. (1990) Non-radioactive labeling and detection of nucleic acids II. Optimization of the digoxigenin system. Mol. Gen. Hoppe-Seyler 371, 929-938. 

An overview on the genosensor technologies for detection of nucleic acids (NA) immobilized onto different transducers by adsorption, cross-linking, complexation and covalent attachment is briefly summarized in Table 19.1. The applications of electrochemical genosensor technology are discussed in the following section. 

H. Xie, C. Zhang and Z. Gao, Amperometric detection of nucleic acid at femtomolar levels with a nucleic acid/electrochemical activator bilayer on gold electrode, Anal. Chem., 76 (2004) 1611-1617. 

In the case of DNA biosensors, two strategies are applied to detect pollutants one is the hybridisation detection of nucleic acid sequences from infectious microorganisms, and the other the monitoring of small pollutants interacting with the immobilised DNA layer (drugs, mutagenic pollutants, etc.) [65]. 

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