Nucleotides, labeled separation

Figure 13.19 The Maxam and Gilbert method of DNA sequencing. The labelled DNA strand is divided into four aliquots. Each is treated to cleave the strand next to a different base resulting in a mixture of different length nucleotides. These nucleotides are separated by polyacrylamide gel electrophoresis and the DNA sequence read from the gel.

Figure 5. Sulfometuron methyl Induces the stringent response. 32p labeled cells of typhlmurlum growing In minimal medium supplemented with valine are untreated (panel A), treated with 100 ym SM (panel B) or treated with 100 ym SM and Isoleuclne (panel C). Extracted nucleotides are separated by two-dimensional TLC and visualized by autoradiography. The upper arrow in each panel indicates the position of ppGpp while the lower arrow indicates the position of pppGpp.

Although in the original method (Breitman, 1963) the counting efficiency was only 2% for (%)-labelled thymidine nucleotides, the technique has also been adopted for assaying thymidine kinase in developing rat cerebellum (Weichsel, 1974). As can be expected, their values are low in comparison with those works in which thymidine nucleotides were separated by thin-layer chromatography (Yamagami et al., 1972). Further-... 

Rabbit liver was perfused with phTj-hypoxanthine, as previously described, followed by washout perfusion for ten minutes with an isotonic solution of salts containing glucose, to remove extracellular label. An oxygenated washed human erythrocyte suspension was then perfused through the liver for 1 hr. Erythrocytes were collected and washed in an isotonic solution of salts. Liver and erythrocyte extracts were chromatographed and assayed. Purine bases, prepared by hydrolysis of the nucleotide fractions, separated by chromatography on Dowex 50. 

Our reviewer felt the molecule builder was easy to use. It is set up for organic molecules. Specialized building modes are available for peptides, nucleotides, and carbohydrates. It is also possible to impose constraints on the molecular geometry. Functions are accessed via a separate window with buttons labeled with abbreviated names. This layout is convenient to use, but not completely self-explanatory. The program is capable of good-quality rendering. At the time of this book s publication, a new three-dimensional graphic user interface called Maestro was under development. 

Biomolecular MS and in particular MALDI-TOF-MS (see Sections 2.1.22 and 2.2.1) permit the routine analysis of oligonucleotides up to 70-mers, intact nucleic acids, and the direct detection of DNA products with no primer labels with an increase in analysis speed and mass accuracy especially in contrast to traditional DNA separation techniques such as slab gels or capillary electrophoresis. Applications focus on the characterization of single nucleotide polymorphisms (SNPs) and short tandem repeats (STRs). Precise and accurate gene expression measurements show relative and absolute numbers of target molecules determined independently of the number of PCR cycles. DNA methylation can be studied quantitatively. 

Fluorescein-labelled deoxyribonucleotide triphosphates may be used in place of those labelled with 32P. Once the DNA sequences are separated by electrophoresis, the resulting DNA bands fluoresce and are analysed by a flu-orogram imager, which produces a picture of the fluorescent bands similar to the autoradiography produced when using 32P-labelled nucleotides. 

Glucose and 0-toluoylglucosamine bind to these crystals in the deep cleft that separates the lobes of each subunit (70). ADP or AMP-PNP only bind in the presence of a sugar substrate or inhibitor. Only one nucleotide binds per dimer, and its binding site is located at the point of contact between the two subunits (72). Parts of the binding site are on each subunit. This site has been labeled the I site (72). A schematic drawing of the BII structure with the location of the various binding sites is shown in Fig. 16 (72). 

The 32P-postlabeling technique allows to improve the sensitivity of the detection of DNA damage (Cadet et al. 1998). The damaged DNA is enzymatically degraded into nucleotide-3-phosphates [reaction (7)]. The resulting mixture of unchanged nucleoside-3-phosphates (dNp) and damaged ones (dXP) are separated by HPLC [reaction (8)]. They are then labeled at the 5 -position with 32P [reaction (9)] and subsequently dephosphorylated at the 3 -position [reaction (10)]. This allows to proceed with a second purification and their identification by, for example, two-dimensional TLC [reactions (11) and (12)]. 

Single-nucleotide polymorphisms (SNPs) have been analysed on a capillary gel electrophoresis (CGE) microchip with EC detection [150]. The genetic section that contained the SNP was amplified by PCR and purified. Then, it was used in a single-base extension (SBE) reaction with a redox-labeled chain terminator, ferrocene-acycloATP. Products of the SBE, ferrocene-labeled SNP and free ferrocene-acycloATP, were separated employing CGE on microchip and detected using sinusoidal voltammetric detection at a pyrolysed photoresist film (PPF) electrode. 

Nagahara, S., et ah (1992). Spin-labeled oligonucleotides site specifically labeled at the internucleotide linkage—Separation of stereoisomeric probes and EPR spectroscopical detection of hybrid formation in solution. Nucleosides Nucleotides 11, 889—901. 

A high-throughput assay for bacterial RNA polymerase has been successfully developed and validated using a 96-well, automated format [70], The reaction mixture contained a DNA template, nucleotide substrates (NTPs), supplemented with a-33P-labeled CTP in Tris-acetate buffer (pH 6.8). The polymerase reaction was carried out at 34°C for 40 min (providing linear kinetics). The effect of dimethylsulfoxide (DMSO), the usual solvent for test compounds used in a screen, was taken into consideration. The radiolabeled RNA transcripts were allowed to bind diethyl aminoethyl (DEAE) beads, which were then separated via filtration, and radioactivity associated with the wells was quantitated to measure the RNA polymerase activity. The standard deviation of the measured activity was typically < 15% of the average. Use of this assay to screen for RNA polymerase inhibitors from chemical libraries and natural products led to the identification of DNA intercalators (known to inhibit RNA polymerase activity), rifampicin (a known inhibitors of RNA polymerase), and several derivatives of rifampicin from Actinomycetes extracts. Therefore this assay can be reliably utilized to detect novel inhibitors of bacterial RNA polymerase. 

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