Digoxigenin, labeled probe detection

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... 

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). 

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... 

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. 

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

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

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). 

For in situ hybridization on plant chromosomes, high quality spreads are needed that are free of cytoplasm and cell wall debris (see Chapters 23 and 24). The methods for chromosome pretreatments, probe denatur-ation, and hybridization and posthybridization washes are described in Chapter 26. This chapter describes the probe hybridization mix for digoxigenin-labeled probes (Section 3.1.) and the methods used to detect the sites of probe hybridization (Sections 3.2. to 3.5.). 

This chapter describes the use of digoxigenin-labeled probes to detect mRNA transcripts in tissue sections. The sites of hybridization of the probe are visualized using an antidigoxigenin antibody alkaline phosphatase conjugate, and a colorimetric reaction. 

A CL ISH assay for the detection of human papillomavirus (HPV) DNA was developed, in which the hybridization reaction was performed using either digoxigenin-, biotin-, or fluorescein-labeled probes [64], The hybrids were visualized using AP as the enzyme label and a highly sensitive 1,2-dioxetane phosphate as chemiluminescent substrate. This assay was applied to biopsy specimens from different pathologies associated with HPV, which had previously proved positive for HPV DNA by polymerase chain reaction (PCR). The analytical sensitivity was assessed using samples of HeLa and CaSki cell lines, whose content in HPV DNA is known (10-50 copies of HPV 18 DNA in HeLa cells and 400-600 copies... 

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... 

Figure 8 Chemiluminescent (A and B) and bioluminescent (C) detections for immobilized hybridizations of PCR product. Dg, digoxigenin Bt, biotin Ad, avidin. Procedure A [30] Biotin moiety is incorporated into PCR products during the amplification reaction, using one 5 -biotinylated primer. The product is hybridized with a Dg-labeled probe and is immobilized on streptavidin-coated magnetic beads. This capture reaction is carried out for 30 min at 37°C. A permanent magnet is used to sediment the beads during washing to remove unbound DNA. By incubation with the washed beads for 45 min at 37°C, anti-Dg antibody conjugated to HRP enzyme is bound to the Dg-labeled probe, and luminol reaction is performed for CL detection. Procedure B [31] Wells of apolystyrene microtiter plate are activated with l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, and then coated with a labeled cDNA probe complementary to an internal region of the target DNA.

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. 

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