Probe radioactively labeled

A useful approach to monitor microbial populations in the biotreatment of hazardous wastes involves the detection of specific sequences of nucleic acids by hybridization with complementary oligonucleotide probes. Radioactive labels, fluorescent labels, and other kinds of labels are attached to the probes to increase sensitivity and simplicity of the hybridization... 

The ability of SPR to probe both kinetic and thermodynamic processes, as well as to provide micro-structural information, make it a very important component of the experimental methodology available to probe molecular interactions occurring at surfaces. Furthermore, it allows some of the limitations of other techniques to be overcome. For example, other methods often require one of the partners to be labelled in some way in order to allow it to be detected. Fluorescent probes, radioactive labels, and attachment of independently detectable molecules (e.g. enzymes) have all been used for this purpose. These suffer from the drawback that they may interfere with the binding of the labelled partner to the unlabelled one, or cause unwanted structural perturbations. SPR observations can be based solely on the dielectric properties of molecules, or their intrinsic light absorption characteristics, and thus require no specific labelling. 

Nucleic acid (deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)) probes utilize labeled, ie, radioactive, enzymatic, or fluorescent, fragments of DNA or RNA (the probe) to detect complimentary DNA or RNA sequences in a sample. Because the probe is tailored for one specific nucleic acid, these assays are highly specific and very sensitive (45). 

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 firagments in the material to be analyzed (DNA, RNA, or protein) are separated by gel electrophoresis. The smaller molecules travel faster and appear nearer the bottom of the gd. The bands of material in the gel are transferred or blotted to the surface of a membrane. The membrane is incubated with a (usually radioactive) labeled probe that will specifically bind to the molecules of interest. Visualization of the labded probe (usually by autoradiography) will reveal which band interacted with the probe. The most common types of blots are compared in Table 1-7-1. Most typically, DNA restriction fragments are analyzed on a Southern blot. 

DNA probes are radioactively labeled single-stranded DNA moleoiles that are able to specifically hybridize (anneal) to particular denatured DNA sequences. Different kinds of probes have been developed for the recognition of particular genes (Table 1-7-2 and Figure 1-7-8). 

The origin of the microarray or biochip can be traced to a seminal publication by Edwin Southern over 30 years ago. Southern described a method by which DNA could be attached to a solid support following electrophoresis and interrogated for sequences of interest by hybridization with a complementary DNA sequence (16). The complementary DNA sequence, termed a probe, was labeled with either a radioactive or a fluorescent marker and hybridized to the DNA target sample, which was immobilized on a sohd support, such as a nitrocellulose filter membrane. 

Total RNA is isolated from the lymphocytes according to standard procedures and used as a template for radioactive labeled cDNA synthesis. The purified cDNA is used as probe for cDNA expression arrays. The advantages of this method as compared to other array systems are as follows (1) Radioactive-labeled probes are more sensitive than fluorescent-labeled probes and therefore need less sample RNA. (2) The primers used in the cDNA synthesis match the genes represented on the array. (3) The primer sequences are longer compared to other array systems, which increases the hybridization fidelity of RNA to the matching correct set of genes and therefore reduces mismatch reactions. 

The radioactive-labeled sample see Note 5) is now denatured by incubating it in a boiling (95-100°C) water bath for 2 min and then transferring it on ice for 2 min. The denatured probe is combined with 15mL prewarmed hybridization solution in a disposable 50- or 15-mL plastic tube and carefully mixed together. 

It is critical that the mixture of hybridization solution and radioactive-labeled probe touches the whole surface of the array and that the movement of the container provides for continuous mixture. 

It is a good idea to expose arrays over a range of 1 to 5 d depending on the age of the radioactive label and the signal strength/quahty of the probe. 

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. 

The probe species is often radioactively labeled, or it may carry a fluorescent tag, or some other chemical or enzymatic moiety to generate a positional signal. For radioactive labeling, a common choice of radioisotope is phosphorus-32 (or 32P), because it can be incorporated as phosphate into DNA or RNA relatively easily, and it emits energetic beta particles that are easy to detect. The radioactivity on the membrane can be used to expose an adjacent x-ray film in a pattern corresponding to the radioactive spots on the membrane. After a suitable exposure time, one develops the film and studies the location and intensity of the images of the radioactive spots to deduce the position and degree of probe hybridization on the membrane. 

Oligonucleotide probe detects Hb S allele. [Note indicates radioactive label.]... 

DNA is denatured and transferred from the agarose gel to a cellulose nitrate sheet. The DNA firmly bound to the sheet is hybridized with a radioactively labeled DNA probe, which carries some of the sequences of interest. The radiolabel, which hybridizes to specific regions of the sheet, is detected by autoradiography. By comparing the results obtained from the DNA of different individuals one can see if the labeled DNAs move with the same or a different mobility. If they move differently, there must be a RFLP difference between the individuals. Detection of an RFLP by this means usually depends on the restriction enzyme used in the initial digestion. Some enzymes show a difference others do not. 

Fig. 2. Binding of a nuclear extract to the ARE probe. A four copy radioactively labelled ARE probe was incubated with a nuclear extract from maize suspension culture cells. From the left the lanes represent free probe probe plus extract probe plus extract competed with a 10-fold excess of unlabelled ARE and, lastly, with a 50-fold excess of unlabelled ARE. The top bands are competed by unlabelled ARE but not by other unlabelled DNAs.

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