Nucleic acid radiolabeled

Enzymes useful for detection purposes in ELISA techniques (Chapter 26) also can be employed in the creation of highly sensitive DNA probes for hybridization assays. The attached enzyme molecule provides detectability for the oligonucleotide through turnover of substrates that can produce chromogenic or fluorescent products. Enzyme-based hybridization assays are perhaps the most common method of nonradioactive detection used in nucleic acid chemistry today. The sensitivity of enzyme-labeled probes can approach or equal that of radiolabeled nucleic acids, thus eliminating the need for radioactivity in most assay systems. 

Methods for Determining Biomarkers of Exposure and Effect. Covalent adducts between reactive carbon tetrachloride metabolites (e.g., the trichloromethyl radical) and cellular proteins, lipids and nucleic acids are known to occur, but at present these can only be measured using radiolabeled carbon tetrachloride. Development of immunological or other methods to detect such adducts in humans exposed to carbon tetrachloride could be of value in estimating past exposures to carbon tetrachloride. 

The use of radiolabeled nucleosides as markers for anticancer activity has become a popular method due to the commercial availability of such compounds. The technique is based upon the knowledge that cells rendered unable to replicate or killed by the anticancer agent are unable to effectively incorporate nucleic acid precursors into their DNA or RNA structure. Therefore, a decrease in cell viability correlates with a decrease in radioactivity relative to a control cell population. Although specific procedures differ, the basic technique involves the incubation of tumor cells in the presence of the radiolabeled compound with or without anticancer agent followed by scintillation counting to determine the radioacti vity of the samples. 

The use of radi olabeled nucleic acid precursors, primarily tritiated thymidine or (deoxy)uridine, however, is subject to numerous drawbacks. Nevertheless, the use of radiolabeled precursors have provided some promising clinical correlation data [174]. 

Although the radiolabeled nucleic acid precursor assays described above produce accurate and reproducible quantification of the number of viable cells in a sample, Mosmann [187] sought to develop a more rapid assay capable of handling large numbers of samples. A colorimetric technique was developed based upon the tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetra-zolium bromide, or MTT. Early studies of MTT by Slater et al. [188] determined that when the MTT tetrazolium salt interacts with the dehydrogenase enzymes... 

In situ hybridization may be defined as the detection of nucleic acids in situ in cells, tissues, chromosomes, and isolated cell organelles. The technique was described in 1969 by two separate groups who demonstrated repetitive riboso-mal sequences in nuclei of Xenopus oocytes using radiolabeled probes (1,2). Refinements in recombinant DNA technology and the development of nonisotopic probe labeling and detection (3) obviate the need for radiation protection and disposal facilities, and have converted nonisotopic in situ hybridization (NISH) from a purely research technique to one that can be used in routine laboratory testing. 

Overall approach. Hepatitis B viral extracts from human subjects are incubated with radiolabelled nucleotides and an active inhibitor. Percent inhibition is calculated based on the amount of de novo viral nucleic acid synthesized with respect to lamivudine as a positive control and phosphate buffer saline (PBS) as a negative control. 

Nucleic acid probes are commonly labeled by enzymatic incorporation of radiolabeled nucleotides or enzymatic addition of radiolabeled phosphate groups to the nucleic acid chain. Proteins, in particular immunoglobulins, are labeled commonly by direct... 

Two relatively new immunodetectable moieties have recently been developed for nucleic acid probe systems which allow us to illustrate two different methods of incorporating labels into nucleic acids. Both methods have been employed for biotin labeling. Originally nucleic acid probes were labeled by enzymatic incorporation of pre-labeled nucleotides, radiolabeled nucleotides, biotin-UTP/dUTP, 5-bromodeoxyuridine (15), or most recently a steroid hapten linked nucleotide analogue, digoxigenin-dUTP (16). 

Separate both samples, along with a radiolabeled nucleic acid size standard, on a high-percentage polyacrylamide gel and expose to film. The radioactive bands present on the gel will indicate the region on the DNA that was protected by the polymerase. 

Fig. 2. Radiolabeling (iodination) of nucleic acids, (a, a ) Plasmid DNA and siRNA concentrations and -radioactivity (CPM/20 gl aliquots) measured in the fractions after purification of the labeled nucleic acids on a Sephadex column G25 PD10. (b, b ) Fraction probes (ca. 1 pg nucleic acid) were electrophoresed (100 V 30 min for sIRNA and 90 min for plasmid DNA) on an EtBr/1% agarose gel in TBE buffer. Lane 1. Unlabeled nucleic acid. Lane 2. (b) Supercoiled DNA Ladder and (b ) peqGold DNA Ladder. Lanes 3,4, and 5. Erections of the J-labeled nucleic acids...

---