Hybridization probes

As the result of high specificity and sensitivity, nucleic acid probes are in direct competition with immunoassay for the analytes of some types of clinical analytes, such as infectious disease testing. Assays are being developed, however, that combine both probe and immunoassay technology. In such hybrid probe—immunoassays, the immunoassay portion detects and amplifies the specific binding of the probe to a nucleic acid. Either the probe per se or probe labeled with a specific compound is detected by the antibody, which in turn is labeled with an enzyme or fluorophore that serves as the basis for detection. 

As of this writing, hybrid probe immunoassays are primarily in the laboratory development stage. Assays using the technology have been developed... 

Hybrid probe—immunoassays are expected to find a specific niche in clinical analysis, especially as a means to adapt probe assays to existing immunoanaly2ers which are locked into a specific enzyme or fluorescence detection technology. Commercialization of the first of these assays is expected by the year 2000. 

Drobniewski E.A., More P.G., Harris G. S. Differentiation of Mycobacterium tuberculosis complex and nontuberculous mycobacterial liquid cultures by using peptide nucleic acid-fluorescence in situ hybridization probes./. Clin. Microbiol. 2000 38 444-447. 

Sotlar K, Escribano L, Landt O, et al One-step detection of c-kit point mutations using peptide nucleic acid-mediated polymerase chain reaction clamping and hybridization probes. Am J Pathol 2003 162 737-746. 

The three main categories of hybridization probes for real-time PCR are (1) cleavage based assays such as TaqMan, (2) displaceable probe assays such as Molecular Beacons and (3) probes which are incorporated directly into primers such as Scorpions. 

Several laboratory tests are available to aid in the diagnosis of gonorrhea and include gram-stained smears, culture, or the DNA hybridization probe. 

Tests used to diagnose C. trachomatis include culture, the enzyme immunoassay, the DNA hybridization probe, or the... 

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... 

Beilin, T. Pulz, M. Matussek, A. Hempen, H.-G. Gunzer, F. Rapid detection of enterohemorrhagic Escherichia coli by real-time PCR with fluorescent hybridization probes. J. Clin. Microbiol. 2001,39, 370-374. 

Kim U.J., Shizuya H., Deaven L., Chen X.N., Korenberg J.R., Simon M.I., Selection of a sublibrary enriched for a chromosome from total human bacterial artificial chromosome library using dna from flow-sorted chromosomes as hybridization probes. Nucl. Acids. Res. 1995 23 1838-1839. 

Forster, A.C., Mclnnes, J.L., Skingle, D.C., and Symons, R.H. (1985) Non-radioactive hybridization probes prepared by the chemical labeling of DNA and RNA with a novel reagent, photobiotin. Nucleic Acid Res. 13, 745-761. 

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. 

Ghosh, S.S., Kao, P.M., McCue, A.W., and Chappelle, H.L. (1990) Use of maleimide-thiol coupling chemistry for efficient syntheses of oligonucleotide-enzyme conjugate hybridization probes. Bioconjugate Chem. 1, 71-76. 

Jablonski, E., Moomaw, E.W., Tullis, R.H., and Ruth, J.L. (1986) Preparation of oligo-deoxynucle-otide-alkaline phosphatase conjugates and their use as hybridization probes. Nucleic Acids Res. 14, 6115-6129. 

Khan, A.M., and Wright, P.J. (1987) Detection of flavivirus RNA in infected cells using photobiotin-labelled hybridization probes./. Virol. Meth. 15, 121-130. 

Reisfeld, A., Rothenberg, J.M., Bayer, E.A., and Wilchek, M. (1987) Nonradioactive hybridization probes prepared by the reaction of biotin hydrazide with DNA. Biochem. Biophys. Res. Comm. 142, 519-526. 

Acridinium esters have also been utilized for chemiluminescent detection of cDNA probes (Fig. 5) [9-11], The hydrolysis rate is much faster when the ester is conjugated to single-stranded DNA, rather than to double-stranded DNA. This means that the chemiluminescence from unhybridized acridinium ester-labeled probe is rapidly lost, whereas the chemiluminescence from the hybridized probe is minimally affected. This permits discrimination between hybridized and unhybridized acridinium ester-labeled DNA probes without separation steps. 

Tests that allow rapid identification of chlamydial antigens in genital secretions are the direct fluorescent antibody test, the enzyme immunoassay (requires just 30 minutes for results), the DNA hybridization probe and nucleic acid amplification tests. 

Acridinium ester—labeled chemiluminescent probes have been utilized to detect the specific protein-coding transcripts and to distinguish between transcripts that code for the 190-kDa protein and the two closely related 210-kDa proteins. The assay is called the hybridization protection assay (D3). In this assay, RNA isolated from the patient s white blood cells is first amplified by PCR. The amplified product is incubated with the chemiluminescent probe. The unhybridized probe is removed by selective hydrolysis in sodium tetraborate buffer, containing surfactant Triton X-100 at pH 8.5, in an incubation step at 60°C for 6 min. After the sample is cooled to room temperature, the chemiluminescence of the hybridized probe is measured in a luminometer. The procedure is reported to detect one leukemic cell in a population of a million or more normal cells. It is also rapid, requiring less than 30 min. Its reliability has been attested to by correlation with results obtained on karyotypic and Southern blot analysis (D3). 

Through a process called hybridization probing, the genes from the samples pair up with their complementary counterparts on the solid supports. When the hybridization step is completed, a scanner (laser beam and camera) is used to capture the fluorescence image of the array. 

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