Non-radioactive labeling

A non-radioactively labeled DNA probe of 742bp was used for the hybridization. The probe was obtained from a genomic DNA fi om FORL r2 using the same oligonucleotide above mentioned and the same conditions. The... 

The design of the photoprobe is based on structure-activity relationship (SAR) studies if available. Ideally the photoprobe should be bioactive over the same range as its parent compound. The next step is to synthesize the bioactive photoprobe in radiolabeled form. Similarly, non-radioactive labels (primarily biotin) can also be attached via a linker arm [18]. 

The non-radioactive labeling utilizes fluorescence, chemiluminescence, or biotin/avidin interactions. Capillary electrophoresis with laser-induced fluorescence was first employed in PAL by Miller et al. [51]. Gilbert and Rando recently reported several biotin-containing heterobifunctional reagents and used them successfully [18] (Fig. 5). 

Fig. 28. Synthesis of labeled DNA probes. A Labeled DNA can be generated using different enzymes (Klenow fragment of DNA polymerase or a terminal transferase) to incorporate labeled nucleotides into specific DNA sequences. Probes can be labeled using radioactive nucleotides or nucleotides labeled with an immunogenic molecule such as biotin. B The labeled probe is then hybridized to the target nucleic acid, which is either bound to a membrane or in a tissue section or cell. An antibody is then used to detect the non-radioactively-labeled probe. C The antibody may be conjugated to a fluorescent or chemiluminescent dye, or an enzyme that produces a color reaction. The target nucleic acid is thus visualized.

Mansfield, E. S. Worley, J. M. McKenzie, S. E. Surrey, S. Rappaport, E. Fortina, P. Nucleic acid detection using non-radioactive labelling methods. Mol. Cell. Probes 1995, 5(3), 145-156. 

For many applications of PNAs (e.g., in hybridization assays or to study cellular uptake) it is necessary to attach a label to the PNA. 8 This can be done with a radioactive or a non-radioactive label. 

Holtke, H.-J., Seibl, R., Burg, J., Miihlegger, K., and Kessler, C. (1990) Non-radioactive labeling and detection of nucleic acids II. Optimization of the digoxigenin system. Mol. Gen. Hoppe-Seyler 371, 929-938. 

Garman, A. J. (1997) Non-radioactive Labelling A Practical Introduction. Academic Press, San Diego, CA. 

Some of these reactions can also be used for non-radioactive labelling (Isaac 1994) since Tag-polymerase, DNA polymerase I and also terminal transferase can accept compounds such as digoxygenin-dUTP (DIG-dUTP) and the dideoxy derivative DIG-ddUTP as substrates. DIG-labelled DNA can be detected colorimetrically by antibodies coupled to peroxidase using chromogenic substrates. Similar procedures can also be used to label RNA using DIG-UTP with T7- or SP6-RNA polymerases. Digoxygenin labelling kits are commercially available. 

Thomas A, Smith HR, Willshaw GA, Rowe B (1991) Non-radioactively labelled polynucleotide and oligonucleotide DNA probes, for selectively detecting Escherichia coli strains producing Vero cytotoxins VTl, VT2 and VT2 variant. Mol Cell Probes 5 129-135 Thompson JS, Hodge DS, Borczyk AA (1990) Rapid biochemical test to identify verocytotoxin-positive strains of Escherichia coli serotype 0157. J Chn Microbiol 28 2165-2168 Tyagi S, Kramer FR (1996) Molecular beacons Probes that fluoresce upon hybridization. Nat Biotechnol 14 303-308... 

Non-radioactive technique based on the use of non-radioactive-labeled nucleic acid probes. 

For nonoligonucleotide probes, in vitro transcription (riboprobes), random-primed labeling, nick translation, and PCR-amplification (all for DNA probes) could be the methods of choice for probe labeling with either radioactive or non-radioactive labels. Unless target sequences are not abundant (neuropeptide mRNAs usually are abundant), the use of nonradioactively labeled probes is highly recommended because of a Speed and ease of detection b. Supenor resolution ... 

Mayr GW (1988) A novel metal-dye detection system permits picomolar-range h.p.l.c. analysis of inositol polyphosphates from non-radioactively labelled cell or tissue specimens. Biochemical Journal 254 585-591. 

The quality assurance and quality control of commercially available radionuclides, non-radioactive labelling kits and ready-to-use radiopharmaceuticals as well as their release are the responsibility of the manufacturer. 

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