Hybridization Assays

A variety of techniques have been developed that take advantage of the ability of single-stranded DNA (or RNA) molecules to pair up with and hybridize to other 

The general procedure is as follows. A sample of nucleic acid, which may contain a particular sequence of interest (the target sequence), is denatured and affixed to a 

FIGURE 3.6 Annealing of two complementary strands of DNA to form a fully paired double-stranded molecule. 

Since hybridization assays are so ubiquitous in recombinant DNA work, it is worthwhile to look at some of the details of the method and some commonly used variants. 

The probe species is often radioactively labeled, or it may carry a fluorescent tag, or some other chemical or enzymatic moiety to generate a positional signal. For radioactive labeling, a common choice of radioisotope is phosphorus-32 (or 32P), because it can be incorporated as phosphate into DNA or RNA relatively easily, and it emits energetic beta particles that are easy to detect. The radioactivity on the membrane can be used to expose an adjacent x-ray film in a pattern corresponding to the radioactive spots on the membrane. After a suitable exposure time, one develops the film and studies the location and intensity of the images of the radioactive spots to deduce the position and degree of probe hybridization on the membrane. 

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Hongmanee P., Stender H., Rasmussen O.F. Evaluation of a fluorescence in situ hybridization assay for differentiation between tuberculous and nontuberculous Mycobacterium species in smears of Low-enstein-Jensen and mycobacteria growth indicator tube cultures using peptide nucleic acid probes. J. Clin. Microbiol. 2001 39 1032-1035. 

Clearly, bDNA is more sensitive than LH, HC, and in-house membrane hybridization assays, but its relatively high LOQ is not optimal for detecting lower titers associated with active liver disease. There have been no differences in clinical specificity among the HBV DNA quantitation methods reported. 

Collins, M. L., et al. (1995). Preparation and characterization of RNA standards for use in quantitative branched-DNA hybridization assays. Anal. Biochem. 226,120-129. 

Hwang, S.-J., et al. (1996). Comparison of three different hybridization assays in the quantitative measurement of serum hepatitis B vims DNA. J. Virol. Methods 62,123-129. 

Kapke, G. F et al. (1997). Comparison of the Chiron quantiplex branched DNA (bDNA) assay and the Abbott genostics solution hybridization assay for quantification of hepatitis B viral DNA. J. Viral Hepat. 4,67-75. 

Patsenker LD, Povrozin YA, Sidorov VI, Tatarets AL, Terpetschnig EA (2009) Fluorescence lifetime based hybridization assay using the new long-wavelength fluorescent label Seta-670. In 24th International conference on photochemistry (ICP 2009). Book of Abstracts, p 430... 

Lanthanides also have potential as DEFRET energy donors. Selvin et al. have reported the use of carbostyril-124 complexes (53) with europium and terbium as sensitizers for cyanine dyes (e.g., (54)) in a variety of immunoassays and DNA hybridization assays.138-140 The advantage of this is that the long lifetime of the lanthanide excited state means than it can transfer its excitation energy to the acceptor over a long distance (up to 100 A) sensitized emission from the acceptor, which occurs at a wavelength where there is minimal interference from residual lanthanide emission, is then measured. 

A similar type of biotin-dendritic multimer also was used to boost sensitivity in DNA microarray detection by 100-fold over that obtainable using traditional avidin-biotin reagent systems (Stears, 2000 Striebel et al., 2004). With this system, a polyvalent biotin dendrimer is able to bind many labeled avidin or streptavidin molecules, which may carry enzymes or fluorescent probes for assay detection. In addition, if the biotinylated dendrimer and the streptavidin detection agent is added at the same time, then at the site of a captured analyte, the biotin-dendrimer conjugates can form huge multi-dendrimer complexes wherein avidin or streptavidin detection reagents bridge between more than one dendrimer. Thus, the use of multivalent biotin-dendrimers can become universal enhancers of DNA hybridization assays or immunoassay procedures. 

Cyanine dyes also are used as labels for oligonucleotide probes. Unlike the hydrophilic cyanine dyes valuable for protein labeling, the use of dye-phosphoramidite compounds to synthesize DNA or RNA probes typically requires the use of more hydrophobic dye structures to make them compatible with the solvents and reactions of oligonucleotide synthesis. Thus, indol cyanines containing few or no sulfonates are used in these applications to label oligos for applications such as array detection, hybridization assays, and RT-PCR. 

Biotin-hydrazide has been used to biotinylate antibodies at their oxidized carbohydrate residues (O Shanessy et al., 1984, 1987 O Shanessy and Quarles, 1985 Hoffman and O Shannessy, 1988), to modify the low-density lipoprotein (LDL) receptor (Wade et al., 1985), to biotinylate nerve growth factor (NGF) (Rosenberg et al., 1986), and to modify cytosine groups in oligonucleotides to produce probes suitable for hybridization assays (Reisfeld et al., 1987) (Chapter 27, Section 2.3). 

Ironically, AP is the enzyme of choice for some applications due to its stability. Since it can withstand the moderately high temperatures associated with hybridization assays better than HRP, AP often is the enzyme of choice for labeling oligonucleotide probes. AP also is capable of maintaining enzymatic activity for extended periods of substrate development. Increased sensitivity can be realized in ELISA procedures by extending the substrate incubation time to hours and sometimes even days. These properties make AP the second most popular choice for antibody-enzyme conjugates (behind HRP), being used in almost 20 percent of all commercial enzyme-linked assays. 

The chemical modification of nucleic acids at specific sites within individual nucleotides or within oligonucleotides allows various labels to be incorporated into DNA or RNA probes. This labeling process can produce conjugates having sensitive detection properties for the localization or quantification of oligo binding to a complementary strand using hybridization assays. 

Reduction of the pyridyl disulfide end after SPDP modification releases the pyridine-2-thione leaving group and generates a terminal—SH group. This procedure allows sulfhydryl-reactive derivatives such as maleimide-activated enzymes (Chapter 26, Section 2.3) to be conjugated with DNA probes for use in hybridization assays (Malcolm and Nicolas, 1984). 

The following protocol is based on the method of Forster et al. (1985). Some optimization may be necessary to obtain the best signal and activity for particular probes in hybridization assays. 

Enzymes useful for detection purposes in ELISA techniques (Chapter 26) also can be employed in the creation of highly sensitive DNA probes for hybridization assays. The attached enzyme molecule provides detectability for the oligonucleotide through turnover of substrates that can produce chromogenic or fluorescent products. Enzyme-based hybridization assays are perhaps the most common method of nonradioactive detection used in nucleic acid chemistry today. The sensitivity of enzyme-labeled probes can approach or equal that of radiolabeled nucleic acids, thus eliminating the need for radioactivity in most assay systems. 

Diamandis, E.P., and Christopoulos, T.K. (1990) Europium chelate labels in time-resolved fluorescence immunoassays and DNA hybridization assays (Review). Anal. Chem. 62, 1149-1157. 

Gruber, M. (2002) FRET compatible long-wavelength labels and their application in immunoassays and hybridization assays. Dissertation, Department of Chemistry and Pharmacy, University of Regensburg, Germany. 

Urdea, M.S., Warner, B.D., Running, J.A., Stempien, M., Clyne, J., and Horn, T. (1988) A comparison of non-radioactive hybridization assay methods using fluorescent, chemiluminescent and enzyme-labeled synthetic oligodeoxyribonucleotide probes. Nucleic Acids Res. 16, 4937-4956. 

Z.P. Aguilar and I. Fritsch, Immobilized enzyme-linked DNA-hybridization assay with electrochemical detection for Cryptosporidium parvum hsp70 mRNA. Anal. Chem. 75, 3890-3897 (2003). 

M. Urban, R. Moller, and W. Fritzsche, A paralleled readout system for an electrical DNA-hybridization assay based on a microstructured electrode array. Rev. Sci. Instrum. 74, 1077-1081 (2003). 

In 1987, CL started to be applied in DNA hybridization assays as an alternative to the use of radioactive tags. These assays are based on the specificity of a binding process that of DNA strands for each other. An unknown DNA can be identified with the Southern blot method in which the strands of the analyte are separated and allowed to interact with labeled probe DNA strands on nitrocellulose filter paper. If the label on the probe is detected, the DNA can be identified and, in some cases, quantitated. Conventionally, radioactive tags were used be-... 

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