Nucleic Acid Hybridization Probes

Ghosh, S.S., Kao, P.M., and Kwoh, D.Y. (1989) Synthesis of 5 -oligonucleotide hydrazide derivatives and their use in preparation of enzyme-nucleic acid hybridization probes. Anal. Biochem. 178, 43-51. 

Davies, D.L. and Clark, M.F. (1994). Maintenance of mycoplasmalike organisms occurring in Pyrus species by micropropagation and their elimination by tetracycline therapy. Plant Pathology, 43 819-823.Davis, R.E., Lee, I.M., Dally, E.L., Delwitt, N. and Douglas, S.M. (1988). Cloned nucleic acid hybridization probes in detection and classification of mycoplasma like organisms (MLOs). Acta Horticulturae, 234 115-122. 

What we present here are examples of what can be done with reference system coding and is not meant to be exhaustive. Particularly, we have omitted areas in states of rapid flux, such as monoclonal antibodies and identification by nucleic acid hybridizations, probes and sequences. These are examples of areas of relative newness and ferment in which coding problems are being seriously investigated internationally in such endeavors as the CODATA/IUIS Hybridoma Data Bank (Bussard et al, 1985) and various gene sequence data banks. 

Marras, S. A. E. Tyagi, S. Kramer, F. R. Real-time assays with molecular beacons and other fluorescent nucleic acid hybridization probes. Clin. Chim. Acta 2006,363, 48-60. 

Nucleic acid hybridization methods use oligonucleotide DNA probes with sequences complementary to a portion of the nucleic acid of the target bacterium38,60 and designed to hybridize with immobilized DNA or RNA on a membrane. After any unbound probe has been washed off, the hybridized probe can be detected.64 66... 

Masuko M, Ohtani H, Ebatal K, Shimadzu A (1998) Optimization of excimer-forming two-probe nucleic acid hybridization method with pyrene as a fluorophore. Nucleic Acids Res 26 5409-5416... 

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... 

W2. Wetmur, J. G., DNA probes Applications of the principles of nucleic acid hybridization. Crit. Rev. Biochem. Mol. Biol. 26, 227-259 (1991). 

Wetmur JG. 1991. DNA probes applications ofthe principles of nucleic acid hybridization. Grit Rev Biochem Mol Biol 26 227. 

The intercalation of polycyclic aromatic compounds into duplex DNA structures was used to develop nucleic acid-based electrochemical sensors.66 For example, the bis-ferrocene-tethered naphthalene diimide (16) was used as a redox-active intercalator to probe DNA hybridization.67 The thiolated probe was assembled on a Au electrode, and the formation of the duplex DNA with the complementary analyte nucleic acid was probed by the intercalation of (16) into the double-stranded nucleic acid structure and by following the voltammetric response of the ferrocene units (Fig. 12.17a). The method enabled the analysis of the target DNA with a sensitivity that corresponded to ca. 1 x 10-20mol. 

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). 

This method provided a simple and fast procedure to identify the presence of a nucleic acid sequence with good sensitivity. On average, the liposomes entrapped several thousand dye molecules, which provided intrinsic amplification of individual binding events. A qualitative measure of nucleic acid hybridization by visualization of the colour on the dip stick was intrinsically provided and quantitative measurements were also possible by use of a reflectometer. This approach resulted in a limit of detection of 1 fmol and a dynamic range of over two orders of magnitude. Maximum signal-to-noise was obtained with 0.2 mol% of probe sequence immobilized on the liposomes. 

Optical nucleic acid biosensors and microarrays based on fluorescence detection make use of fluorescent dyes to provide a measurable signal for target/probe hybridization. Ideally, fluorophores used for detecting nucleic acid hybridization should exhibit large molar absorptivity, resistance to pho-tobleaching, quantum yields that approach unity and the abibty to produce a resolvable signal at low concentrations both quickly and reproducibly [40]. 

Solution-based nucleic acid hybridization assays represent a class of analytical methodologies that provide for detection of target-probe hybridization events. Solution-based assays offer several advantages in comparison to configurations that use surface-bound probes, primarily from the standpoint of negating the requirement for immobilization of a probe sequence to a solid surface. Therefore, thermodynamic, kinetic and adsorptive effects that are relevant in terms of consideration of hybridization at interfaces are not encountered. Solution-based hybridization assays provide a simple way of detecting hybridization events in real-time. 

Peptide Nucleic Acids Robust Probe Hybridization Technology... 

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