Digoxigenin labeling hybridization

In the method shown in Figure 9A, a biotin-labeled cDNA probe is first immobilized to a polyvinylchloride microtiter plate well that is coated with bio-tinylated-bovine serum albumin [33], The target DNA is hybridized in the liquid-phase with a digoxigenin-labeled probe, so that the biotin-labeled probe can capture a marker enzyme. An antibody-conjugated enzyme is then added, followed by a chemiluminescent substrate. 

Meltzer JC, Sanders V, Grimm PC, Stern E, Rivier C, et al. 1998. Production of digoxigenin-labelled RNA probes and the detection of cytokine mRNA in rat spleen and brain by in situ hybridization. Brain Res Prot 2 339-351. 

Tim, E. A. and Stewart, C. C. (1992) Eluorescence in situ hybridization in suspension (FISHES), using digoxigenin-labeled probes and flow cytometry. Biotechniques 12, 362-367. 

In the tissue sections, a hybridization is performed with a highly specific probe in order to detect the target (either amplified by indirect in situ RT-PCR or unamplified). For the preparation of nonradioactive digoxigenin-labeled probes, one should refer to the guidelines of the company manuals Genius M System User s guide for membrane hybridization and Nonradioactive in situ hybridization application manual (2nd ed., Boehringer Mannheim). 

Add 2 ng/pL digoxigenin-labeled oligo probe into the following hybridization solution 50% deionized formamide, 10 mL 20X SSC, lOX (final concentration) lOOX Denhardt solution, 1 mg/mL (final concentration) 10 mg/mL salmon sperm DNA, 1% (final concentration) 10% SDS. 

Morey, A L, Ferguson, D J P, Leslie, K O., Taatses, D. J, and Fleming, K. A (1993) Detection of parvoviral and human DNA sequences at the ultrastructural level by in situ hybridization with digoxigenin-labeled probes. Histochem J 25,421-429... 

Fig. 1. Dual nucleic acid detection. The target DNAs (A and B) are denatured and hybridized with complementary DNA probes labeled with biotin (Bio ), or digoxigenm (Dig ). Biotin and digoxigenm residues are detected, respectively, with avidin peroxidase (red) and antibody to digoxigenin labeled with alkaline phosphatase (blue).

Digoxigenin-labeled chromosome 10 a-satellite probe (DIOZI Oncor, Gaithersburg, MD) is used at a final concentration of 5 pl/100 pi in hybridization buffer (50% formamide, 10% dextran sulfate, 0.004% Tween 20, and standard saline citrate (SSC) [in 1.5 strength]). A 10-pl volume of the probe is placed on an 18 x 18-mm coverslip, which is placed onto the slide, sealed with special Vulcanizing Fluid, and placed on a flat-bed thermal cycler. Slides are incubated for 3 min at 94°C and placed in a humidified box for 16 hr at 37°C. 

The genosensor design based on Av-GEB not only is able to successfully immobilize onto the electrode surface the mecA biotin-labelled capture probe, while the hybridization with the mecA target and the mecA digoxigenin-labelled probe is occurring at the same time, but also is capable of distinguishing SNPs [58,65]. 

Hilton, D. A., Love, S., and Barber, R. (1997) Demonstration of apoptotic cells in tissue sections by in situ hybridization using digoxigenin-labelled poly(A) oligonucleotide probes to detect thymidine-rich DNA sequences. J. Histochem. 

Methods have been developed to detect ASOs in plasma and other biological samples, based on hybridization with labeled complementary probes [89], The probes are tagged at one end with biotin and the other end with digoxigenin. After hybridization and binding to neutravidin-coated 96-well plates, nuclease SI is added to degrade unhybridized probe. Anti-digoxigenin antibodies and enzyme-linked secondary antibodies are then added sequentially, followed by enzyme substrate (for example, AttoPhos, which fluoresces after enzymatic cleavage) for detection and quantitation [60, 61]. 

---