Digoxigenin chemiluminescent detection

Chapters 17-22 describe the hybridization of the nonradioactive probes to the DNA and RNA immobilized on blots, together with the detection systems necessary to reveal where the probe has hybridized. Chapters 17-19 deal with digoxigenin probes, with Chapters 17 and 19 describing chemiluminescent detection on DNA and RNA blots respectively, and Chapter 18 describing a colorimetric detection system. Chapter 20 deals with enhanced chemiluminescent detection of enzymically labeled probes, whereas Chapters 21 and 22 describe enhanced chemiluminescent detection of large (Chapter 21) and small (oligonucleotide. Chapter 22) probes labeled with fluorescein. 

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... 

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.

PCR can be used to introduce labels that can then be used for detection. The ability to add to the 5 end of the primers sequences not complementary to the target template, which then becomes incorporated into the double-stranded PCR product, allows the introduction of labels. Thus, the addition of biotin to the 5 end of the primer allows detection of hybridized PCR product with streptavidin or avidin-enzyme conjugates (B4). Other labels such as digoxigenin can be added to the 5 end of the primer, amplified, and detected either colorimetrically or by chemiluminescence (F3). 

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... 

LM Weigel, JT Belisle, JD Radolf, MV Norgard. Digoxigenin-ampicillin conjugate for detection of penicillin-binding proteins by chemiluminescence. Antimicrob Agents Chemother 38 330-336, 1994. 

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

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