Radio-labeled probes

Incubate >1 /ig of RNA with a 3- to 10-fold molar excess of radio-labeled probe (about 150 to 600 pg per 10 /ig total RNA) in hybridization buffer. 

However, if the gene has been cloned, but the required activity is not produced, then the functional test will fail to pick up the target gene. In this case, if some gene sequence information is available, then it may be possible to test for the presence of DNA with the expected sequence by hybridization with radio-labelled probe DNA or, more usually, by PCR. This sequence-based screening test could pick up positives which have been missed in the initial screen because the gene has been successfully cloned but the enzyme has not been produced in an active form (perhaps because expression has not occurred or because E. coli is a poor host to support production of active enzyme), or where there is no convenient function-based assay available. 

The original colony hybridization method described the use of radio-labeled probes and the detection of positive hybridization events by autoradiography (1). However, because of the high waste disposal costs, short half-lives, long autoradiographic exposures, and potential health hazards associated with radioisotopes, there is interest in alternative methods to detect positive hybridizations. 

M. Nickho-Amiry, J.M. Winfield, Investigation of fluorinated surfaces by means of radio-labelled probe molecules, J. Fluorine Chem. 128 (2007) 344-352... 

Lee CC, Sui GD, Elizarov A, Shu CYJ, Shin YS, Dooley AN, Huang J, Dari-don A, Wyatt P, Stout D, Kolb HC, Witte ON, Satyamurthy N, Heath JR, Phelps ME, Quake SR, Tseng HR (2005) Multistep Synthesis of a Radio-labeled Imaging Probe Using Integrated Microfluidics. Science 310 1793— 1796... 

DNA arrays have been categorized into different formats based upon what is immobilized to the surface (also known as the solid phase, substrate, or chip) and what is captured from the sample solution. Definitions change depending upon the format. For the classic Southern dot blot, the sample was first spotted down on the surface, cross-linked, and then bathed with a radio-labeled oligonucleotide under hybridization (complementary nucleic acid strand base-pairing) conditions to detect the presence of a parhcular sequence within the sample. This was called probing. The oligonucleohde... 

Using radio-labeled thiamine probes, a study of thiamine metabolism at normal loads produced an estimated half-life of thiamine of 9.5 to 18.5 days, and showed a large number of breakdown products in the urine. Several of these urinary catabolites are shown in Figure 30-9. 

Radio-labeled single stranded DNA oligonucleotide probes are allowed to anneal with the target strand in an oligonucleotide hybridization analyses. 

TTot atom radio labeling processes often leave primary products with large amounts of internal excitation. As a consequence, secondary unimolecular fragmentations can play an important, if not dominant, role in the chemical systematics of nuclear recoil activated compounds. This particular aspect of hot atom chemistry has been used to advantage by a number of workers who have applied nuclear recoil chemical activation to probe the kinetics n dyn jcs j J un q ecular rate processes (i-I5). 

The most commonly used radioisotopes for PET are listed in Table 13.1. Since their half-lives are on the order of tenths of minutes, an important challenge in PET imaging is to develop efficient and fast chemical processes to synthesize the radio-actively labelled molecules of interest. The short half-lives of the radioisotopes used in PET represent an important limitation of the technique since the labelled probes must often be prepared in a cyclotron in close proximity to the PET instrument. 

Figure 6 Schematic of the first RIA, for insulin [15]. (a) Polystyrene spheres with anti-insulin antibody immobilized on surface, (b) Sample matrix containing analyte and interferents, together with radiolabelled insulin probe, are added, (c) Analyte insulin and probe radio-labelled insulin compete for antibody binding sites, (d) Matrix containing interferents, unbound analyte and unbound probe, is discarded, (e) Radioactivity of polystyrene spheres is recorded. The more anal de present in the sample, the less radioactivity is recorded.

---