Oligonucleotide probe chemiluminescent-labeled

The second technique uses a homogeneous assay format with no physical separation between hybridized and nonhybridized probes. The method is the result of a discovery that for a suitably positioned acridinium ester, within an oligonucleotide sequence, hybridization prevents hydrolytic attack on the ester by hydroxide ions. Such hydrolysis would lead to the inactivation of the chemiluminescent label (see Section 3.2.1). This differential susceptibility to hydrolysis forms the basis for a so-called hybridization-protection assay (HPA), which is schematically illustrated in Fig. 30. 

Hybridization of Fluorescein-Labeled Oligonucleotide Probes and Enhanced Chemiluminescence Detection... 

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). 

An elegant approach is to capture the target DNA or RNA with specific oligonucleotides on to a microwell plate. Synthetic branched DNA bearing multiple alkaline phosphatase-labeled probes hybridizes to the target. A chemiluminescent substrate is added to produce signal. This branched DNA assay has been used in infectious disease detection (W3). 

Chapters 17-22 describe the hybridization of the nonradioactive probes to the DNA and RNA immobilized on blots, together with the detection systems necessary to reveal where the probe has hybridized. Chapters 17-19 deal with digoxigenin probes, with Chapters 17 and 19 describing chemiluminescent detection on DNA and RNA blots respectively, and Chapter 18 describing a colorimetric detection system. Chapter 20 deals with enhanced chemiluminescent detection of enzymically labeled probes, whereas Chapters 21 and 22 describe enhanced chemiluminescent detection of large (Chapter 21) and small (oligonucleotide. Chapter 22) probes labeled with fluorescein. 

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