Digoxigenin-labeled probe

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... 

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). 

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... 

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)... 

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. 

Hopfenbeck JA, Holden JA, Wittwer CT, Kjeldsberg CR. Digoxigenin-labeled probes amplified from genomic DNA detect T-cell gene rearrangements. Am f Clin Pathol 1992 97 638-44. 

Gentilomi G, Zerbini M, Musiani M, et al. In situ detection of B19 DNA in bone marrow of immunodeficient patients using a digoxigenin-labelled probe. Mol Cell Probes. 1993 7 19-24. 

Digoxigenin-labeled probes are typically quite stable and may be stored at -20°C for several months with no apparent loss sensitivity. Radiolabeled probes typically show sufficient activity to be used over several days in the range of about 1 wk. 

Ch, 29. Detecting mRNA in Tissue Sections with Digoxigenin-Labeled Probes,... 

Until recently, the hybridization probe has always required radioisotopic labeling (normally with P), particularly if the genome under study was derived from a eukaryote. This placed artificial limits on the number of probes that could be manipulated by any laboratory within a set time period, and had a major impact on staff training, laboratory layout, laboratory access, and management. The recently developed nonradioactive systems (e.g., digoxigenin-labeled probe... 

Hybridization of Digoxigenin-Labeled Probes to Southern Blots and Detection by Chemiluminescence... 

Incubate the blots in approx 200 mL of antidigoxigenin solution for 30 min. At this point m the process the antibody/alkaline phosphatase conjugate binds to the digoxigenin-labeled probe that is bound to the membrane. 

Fig. 1. Southern hybridization with a digoxigenin labeled probe. This is a lumi nogram of a unique sequence probe labeled with 5% digoxigenin modified base and hybridized to a maize genomic Southern as described. With a 3-h exposure at 37°C single copy fragments are readily detectable.

The following procedure describes the hybridization of digoxigenin-labeled RNA minisatellite probes see Chapter 12) to Southern blots of different species and visualization of the DNA banding patterns by color detection. The method can be applied to both multi- and single-locus probes see Figs. 1 and 2, respectively) and also for blots that have been screened several times with both radioactively labeled or digoxigenin-labeled probes (1). 

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