Digoxigenin labeling

A CL ISH assay for simultaneous detection of two different viral DNAs (HSV and CMV DNAs) was developed utilizing both HRP and AP as reporter enzymes [63], A biotinylated HSV DNA probe and a digoxigenin-labeled CMV DNA probe were cohybridized with samples then CL detection of the two probes was performed. The HSV DNA was revealed using a streptavidin-HRP complex... 

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. 

In the method shown in Figure 9B, a firefly luciferase gene is introduced for sensitive bioluminescent detection of target DNA [5], The luciferase-coding DNA requires no posttranslational modification, and the activity of the luciferase produced can be readily measured in the transcription/translation mixture without prior purification. In this assay system, the digoxigenin-labeled probe is first immobilized to polystyrene wells coated with antidigoxigenin antibody. The target... 

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). 

In the case of direct in situ RT-PCR, digoxigenin-labeled 11-dUTP is added in the 4(dNTP) mix. The recommended concentration ratio of digoxigenin 11-dUTP to dTTP is 1 19 (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. 

Incubate with the biotin or digoxigenin-labeled lectin, diluted in Soln. A to 5-50 pg/ml, for 1 h at RT. Wash at least three times with PBS or TBS. 

Hybridise both fluorescein and digoxigenin-labelled probes 2.1ucubiiie wim tut li-fluoresce in hurserudish peroxidase 3.Slain with Fast Red... 

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... 

In addition to biotin, a digoxigenylated derivative of dUTP was also synthesized. This derivative of dUTP can be incorporated into DNA by Pol I (or the Klenow fragment of Pol I). Therefore, digoxigenin-labeled DNA probes can be prepared by nick translation or random primed-labeling methods developed for the biotin system. It is almost certain that more nonradioactive alternatives to biotin and digoxigenin will be developed in the future. Chemiluminescent methods for nonradioactive probe detection are now widely being used... 

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).

Biotin- and/or digoxigenin-labeled probes are stable for more than a year when stored at 4°C It is therefore possible to nick translate up to 5 pg of the probes and store a good batch. When compared with tritium (5 ng), the amount of probe used is much larger (typically 20-100 ng)... 

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]. 

Hemngton, C. S., Bums,J, Graham, A. K, Evans, M. F., and McGee, J. O D. (1989) Interphase cytogeneucs using bioun and digoxigenin labeled probes 1 Relauve sensitivity of both reporters for detection of HPV16 in CaSki cells./. Clin. PathoL 42, 592-600. 

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