Radioactive probes

Nick translation A technique for labeling DNA based on the ability of the DNA polymerase from E colt to degrade a strand of DNA that has been nicked and then to resynthesize the strand if a radioactive nucleoside triphosphate is employed, the rebuilt strand becomes labeled and can be used as a radioactive probe. 

Prepare a radioactive probe to hybridize with the membrane. 

PET has been gaining in popularity as a result of its enhanced sensitivity and resolution with respect to SPECT at the millimetre scale, allowing clinicians to study drug distribution in vivo (10). Indeed, it is estimated that by 2010 there will be approximately two million PET scans per year, of which the majority will image the metabolism of the 18F-labeled fluoro-deoxyglucose (FDG) (11). The increased sensitivity in turn reduces the concentration of the administered radiopharmaceutical required to 10 8-10 10M. The much lower concentrations of administered radioactive probe employed reduce the overall... 

The collection of colonies produced is referred to as a genomic DNA library. The library must be screened with a radioactive probe to identify the colony with the desired restriction fragment (see Screening DNA Libraries). 

Colonies on the blot are lysed and treated with a radioactive probe specific for the DNA sequence ( P-DNA) or recombinant protein ( 1-antibody reactive with the recombinant protein). 

For nonradioactive probe flnorescence or enzymatic color development For radioactive probe dip in photographic emulsion, expose, develop, stain Microscopic examination... 

Cellular autoradiography techniques using radioactive nucleic acid probes have several features in common with nucleic acid immunocytochemistry. The method is based on the hybridization of radioactive probes to cellular targets and the subsequent exposure of photographic emulsion, which, when developed, reveals blackened (exposed) silver grains close to the site of hybridiza-hon. Hence, cellular autoradiography techniques permit excellent specihcity and localizahon of the hybridized probe—to 1 qm when tritium is the label used in the autoradiography-based method (9). 

Fig. 4. Illustration of the multiplex allele specific diagnostic assay. At the top of the panel, radioactive ohgonucleotide probes (indicated by stars) are selected by hybridization to amphcons from patient samples affixed to membrane filters. When a putative mutation-bearing allele hybridizes to a radioactive probe, it can be eluted from the filter and subjected to sequencing using chemical or radioactive dideoxy-terminator methods. This permits unequivocal identification of a large number of mutations at a high throughput.

Regional Cerebral Blood Flow Mapping. Such mapping techniques use radioactive probes (e.g., xenon-13) to delineate perfusion of cortical structures. For example, they have generally confirmed prefrontal cortex dysfunction in schizophrenia ( 37). 

Radioactive probe hybridizes to restriction fragments A, B, and C, producing dark bands on exposed x-ray film. 

The products of gene expression (mRNA and proteins) can be measured by techniques such as the following. Northern blots are very similar to Southern blots except that the original sample contains a mixture of mRNA molecules that are separated by electrophoresis, then hybridized to a radioactive probe. Microarrays are used to determine the differing patterns of gene expression in two different types of cells—for example, normal and cancer cells. Enzyme-linked immunosorbent assays (ELISAs) and western blots (immunoblots) are used to detect specific proteins. 

The higher the photon energy, the smaller are in general absorption coefficients and the less severe are absorption problems, which simplifies the design of experiments. If radioactive (probe) nuclei can be embedded into the sample crystal (as is the case with PAC), no external radiation is needed for the investigation of solid state... 

Another procedure for amplification cuts DNA containing the segment of interest into small pieces with a restriction enzyme. The cut pieces are incorporated into a plasmid or virus vector to be amplified in a suitable host. After growth, the mixture is plated to produce a mixture of bacterial or viral clones. The clone or clones of interest are identified often by hybridization of the clones after replica plating with a radioactive probe, followed by autoradiography to find the clone of interest. 

A more satisfactory procedure involves a radioactive probe held a little above the water surface. The radioactive radiations ionise the air between the probe and the surface and ensure that they are at the same potential. The probe is connected to an electrometer having a very high input impedance which reads the surface potential. This procedure was pioneered by Guyot (70J and Frumkin [71J in the 1920s but has become much more convenient to use with the introduction of artificial radioactive isotopes and modern electronics. Americium 241 is particularly useful as the low energies of the a and y radiations produced only ionise the air in the immediate vicinity of the probe. 

The controversy over the degree to which radioactive probes are more sensitive has not been fully resolved. In any case, microwave pretreatment enhances ISH signal detection of RNA and DNA whether radiolabeled or nonradioactive probes are used both methods are presented later. Furthermore, a number of approaches is available to increase the sensitivity of the nonradioactive ISH procedures (for a review, see Komminoth and Werner, 1997) some of these approaches are discussed below. 

In both Southern and Northern analyses restriction-digested DNA fragments, mRNA, and polyA mRNA are separated by size when electrophoresed on agarose gel. The separated molecules are transferred, by electroblotting or capillary blotting, on to a nylon or nitrocellulose membrane. The immobilized RNA or DNA is reacted with a radiolabeled, chemiluminescent, or fluorescent probe that is complementary to the DNA/RNA of interest, unbound probe is washed off, and the membrane exposed, in the case of radioactive probes, to radioautographic film to visualize the sample of interest. 

Figure 15.2 Southern blotting. The exposed portion of the film shows the DNA fragment complementary to the radioactive probe.

The concentration of the sample can be estimated by examining the ethidium bromide gel or by using a spectrophotometer. The products from this particular preparation can then be used for RFLP with end labeling, for cloning, for PCR, or for making radioactive probes for Southern blots or filter lifts. The purity of the sample is essential for all applications except PCR. 

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