Nucleic assay formats

While the capture on DNA chips of fluorophore-labelled targets, and the extension of arrayed primers with fluorophore-labelled nucleotides has been widely used for some time, it is only more recently that assay formats have developed that utilize immobilized nucleic acids already modified with fluorophores. Fundamental analyses of surface monolayer structures and chemistries can be readily performed by immobilizing such modified oligonucleotides into SAM structures [105,106], but it is those interactions that can be monitored using fluorescence quenching or fluorescence resonance energy transfer (FRET) that have gained the most attention. 

Assay Formats Two main assay formats are used in lateral-flow assays the competitive assay and the sandwich assay format. Both formats have been used for nucleic acid lateral flow assays, though the latter is more common (56, 58). The competitive assay format utilizes an analyte... 

Nelson, N. C., Hammond, P. W., Weise, W. A., and Arnold, L. J., Jr., Homogeneous and heterogeneous chemiluminescent DNA probe-based assay formats for the rapid and sensitive detection of target nucleic acids. In Luminescence Immunoassay and Molecular Applications (K. Van Dyke and R. Van Dyke, eds.), pp. 293-309. CRC Press, Boca Raton, FL, 1990. Nelson, R., Jiang, N.-S., and Shellum, C., A simple and sensitive immunochemiluminescent assay for tumor necrosis factor-alpha. Ctin. Chem. (Winston-Satem, N.C.) 38, 1079-1080 (abstr) (1992). 

Only limited development of new methodologies has taken place for immunochemical analysis of nucleic acids. Most published methods rely on modifications to classical DNA probe hybridization or immunoassay methods, with considerable blending of the two. For example, some methods employ immobilized oligonucleotide probes to capture the analyte DNA followed by immunoenzymatic detection. Other methods use immunocapture followed by detection with an enzyme-labeled DNA probe. Distinctly new methodologies mostly impact on assay formats (e.g., DNA microarrays and in situ hybridization) and detection reagents (e.g., chemiluminescent enzyme substrates). 

Assay Formats for Nucleic Acid Analysis by MALDI-MS... 

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). 

In this chapter, we will survey the kinds of solid supports (substrates) and surface chemistries currently used in the creation of nucleic acid and protein microarrays. Which are the best supports and methods of attachment for nucleic acids or proteins Does it make sense to use the same attachment chemistry or substrate format for these biomolecules In order to begin to understand these kinds of questions, it is important to briefly review how such biomolecules were attached in the past to other solid supports such as affinity chromatography media, membranes, and enzyme-linked immxm-osorbent assay (ELISA) microtiter plates. However, the microarray substrate does not share certain unique properties and metrics with its predecessors. Principal among these are printing, spot morphology, and image analysis they are the subjects of subsequent chapters. 

As with other analytical platforms, there are some drawbacks associated with the use of MALDI-TOF MS for DNA analysis. Nucleic acids are susceptible to adduct formation and fragmentation. Their negatively charged backbone makes them behave as polyanions in aqueous solutions. In the presence of cations they tend to form adducts. Most enzymes used in biochemical assays require cations as co-factors, and the predominant ones are sodium (23 Da), magnesium (24 Da), and potassium (39 Da). For example, a sodium ion would add 22 Da to the mass of the analyte, since it would replace a proton (1 Da). Therefore, if adducts are not removed prior to analysis, a distribution of several signals may be obtained, resulting in lower... 

Fig. 5 Immobilized nucleic acid assays utilizing redox-active moieties, a Amplified detection of viral DNA by generation of a redox-active replica and the bioelectrocatalyzed oxidation of glucose (Reprinted with permission from [200]. Copyright(2002) American Chemical Society), b Alternative formats for the capture on a gold electrode SAM of solution-extended primers or direct surface extension of primer with electrotides (adapted from [185]). c Ferrocene-labelled hairpin for electrochemical DNA hybridization detection. A Fc-hairpin-SH macromolecule is immobilized on a gold electrode. When a complementary DNA target strand binds to the hairpin, it opens and the ferrocene redox probe is separated from the electrode, producing a decrease in the observed current (Reprinted with permission from [203], Copyright(2004) American Chemical Society)...

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