Nucleotides, labeled

DNA. A synthesis of the complementary strand with nucleotides labeled with fluoro-phores. B attachment of this strand to a... 

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.

The main problem in this approach is the very low permeability of mevalonic acid to membranes, resulting in very low incorporation. Positive results have been obtained by the use of cell-free systems incubated with [14C]-mevalonic acid,26,27 [14C]isopentenyl diphosphate,28 or [32P]orthophos-phate.29 Incubation of these radioactive lipids with glycosyl nucleotides labelled in the glycosyl group with a different isotope, followed by extraction and cochromatography in different solvent systems, may indicate that both compounds are present in the same molecule. When the lipid moiety becomes labelled from mevalonic acid or isopentenyl diphosphate, chromatography on DEAE-cellulose columns should be performed, in order to avoid confusion with steryl glycosides. 

Introducing in the 5 -position a T-containing nucleotide labeled with deuterium at either C(l )> C(2 ) or C(4 ) showed only a marked kinetic isotope effect (KIE = 3.9) when labeled at C(l ). It has been concluded that the observed effects must be due to an H-abstraction of the Thy-5-peroxyl radical from the neighboring C(l ) position. A kinetic isotope effect is only observed when there is a competing reaction. This competing reaction has not yet been identified. However, some information has been obtained by generating specifically the C(l ) radical within a short ODN [reactions (11) and (12) Hwang et al. 1999],... 

Fig. 8.7 The principle of full genome sequencing using labelled dideoxy (dd) nucleotides labelled fragments are eluted through a gel and detected in order of length to determine the complementary DNA sequence from which the original can be deduced...

Fig. S. In vitro transcription of a cloned DNA fragment from Anabaena azollae containing a self-splicing intron. (A) Restriction map of a 2.7-kb insert in pBSM13—. Arrows indicate direction of transcription from the T3 and T7 promoters in the vector. tRNA exon (solid) and intron (hatched) sequences are indicated. (B) Plasmid DNA truncated by Ps/I (P), SlpI (S), Dral (D), and Hindi) (H) (in the 3 exon), downstream of the T3 promoter. After transcription with T3 RNA polymerase, products were fractionated on a 3% polyacrylamide-8 M urea gel the autoradiogram is shown. Scale at left indicates position of Haelll restriction fragments of phage 0X174 DNA in nucleotides. Labels indicate positions expected for the unspliced run-off transcript (Pre), ligated exons (LE), and linear intron (LI). (From Xu et at. Copyright 1990 by the AAAS.)...

A useful procedure for estimating adenylate cyclase in intact cells and tissues is to incubate the tissue with labelled adenine and then measure the rate of labelling of cyclic AMP [112]. Adenine readily penetrates cells and is partially converted to ATP. In heart slices, the ATP newly synthesised from radioactive adenine was found in equilibrium with the existing pool used for the production of cyclic AMP the specific activity of the newly-formed cyclic AMP was similar in the presence and in the absence of stimulatory hormone [113]. The prelabelling method has been compared with the protein-binding method in brain slices [114,115]. Increases in total levels of cyclic AMP and increases in levels of radioactive cyclic AMP derived from intracellular adenine nucleotides labelled by prior incubation with radioactive adenine occurred on similar time courses and to similar extents. Radioactive cyclic AMP represented a small (7-13%) but relatively constant fraction of the total amount of cyclic AMP. These results provided no evidence for the presence of more than one major compartment of adenine nucleotides in brain slices that serve as a source of nucleotide precursor for cyclic AMP. The nucleotides of this compartment were uniformly labelled by incubation with radioactive adenine [116]. 

The nick-translation method was originally used to incorporate radiolabeled nucleotides into DNA probes. More recent work has shown that nucleotides labeled with biotin can also be incorporated using this method, to yield probes of equivalent sensitivity. Uracil and adenine nucleotides that have been labeled with biotin are shown in Figure 9.18. 

Preparation of fluoranthene-modified DNA in vitro digestion with enzyme isolation of adducts using disposable C g cartridge nuclease P1 pretreatment to remove residual unmodified nucleotides labelling of fluoranthene-DNA adduct by postlabelling technique nuclease P1 digestion... 

By use of n-glucosyl nucleotides labeled with carbon-14 in the n-glucosyl moiety, it was found that the uridine, 2-deoxyuridine, or thymidine 5-(a-D-glucopyranosyl pyrophosphates) could serve equally well as n-glucopyranosyl donors in the first reaction. This apparent lack of specificity for the pyrimidine portion of the base might be due to the existence of one nonspecific enzyme or to a mixture of several specific enzymes. 

The low RBC ATP concentration is the most probable explanation for the hemolysis (1). In vitro studies of adenosine metabolism by intact patient s RBC showed as expected, a markedly decreased ATP synthesis from adenosine moreover, metabolic studies of adenylic nucleotides labelled with radioactive adenine indicate that AMP degradation (probably by hydrolysis of the phosphate ester followed by deamination of adenosine) is abnormally elevated in the patient s erythrocytes. Thus, the low RBC ATP concentration appears to be secondary to both a diminished synthesis of AMP from adenosine and an excessive catabolism of AMP. 

A DNA molecule is a long chain-like chemical consisting of four units called nucleotides labeled as A, C, G, and T. You may visualize it as, say, a necklace made of beads of four different colors, aquamarine (A), green (G), cobalt blue (C), and tan (T) (Fig. 4.1a). A necklace may consist of at most hundreds of beads, but a DNA molecule may be made of hundreds of thousands or even millions of these... 

Figure 10 incorporation into hydroxypyridone nucleotide-labeled DNA forms Cu... 

The 3 -end labeling is typically carried out using terminal deoxynucleotidyltrans-ferase in the presence of P- or S-labeled ddNTP, 3 -deoxy-ATP (cordycepin triphosphate), or rATP. (Note For most of the procedures involving radiolabeling, nucleotides labeled with should be considered as a preferred substitute.)... 

In an extension of the erythrocyte perfusion experiment, a liver labeled by hypoxanthine perfusion, was removed from the rabbit and slices were prepared. The labeled liver slices were incubated for one hour in suspensions of washed human and rabbit erythrocytes, in vitro. The results, presented in Table III, indicate that as in the in situ experiment, over 80 percent of the nucleotide label in the human erythrocyte was present in the adenine nucleotides, undoubtedly derived from adenosine released from the liver cells. It is interesting to note that a similar distribution of label appeared in the... 

Houthoff, H. J. Reedijk, J. Jelsma, T. Heetebrij, R. J. Volkers, H. H. Methods for labeling nucleotides, labeled nucleotides and useful intermediates. Application Information W0 977NL559 19971008... 

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