Oligonucleotide samples, labeling

Availability of a sample isotopically enriched with 15N and 13C offers the NMR spectro-scopist additional tools for overcoming the problems encountered in homonuclear experiments. In an obvious extension of the homonuclear experiments, the carbon or nitrogen dimension can be used to improve the resolution in NOESY spectra. However, recent trends clearly favor the through-bond approach where the presence of a cross peak unambiguously proves the existence of a chemical bond between the nuclei involved. Let us review briefly the experiments available for labeled oligonucleotides. 

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... 

The oligonucleotide samples described here were labeled at the 5 end according to the following procedure. Approximately 0.5 OD of oligomer sample was combined with 5 j L of 7- P ATP (10 mCi/ml, 5000 ci/mmole, Amersham), 5 j L of lOx kinase reaction buffer... 

Sample Preparation. All samples were prepared for SERRS analysis using the following amounts of reagents, 10 pi of dye-labeled oligonucleotide, 10 pi of 0.1 mol dm spermine, 250 pi of water, and 250 pi of citrate reduced silver nanoparticles. 

Fig. 5.22. A DNA array is an orderly arrangement of immobilised oligonucleotides on a glass slide, each grey spot represents a different oligonucletide. When reacted with labelled DNA samples, they hybridise with only certain spots on the array, i.e. those containing a matching oligonucleotide sequence. This results in a characteristic pattern, a fingerprint, of coloured and uncoloured spots.

Fig. 19. Fluorescence image of an array of oligonucleotide probes following hybridization of a fluorescently labeled RNA sample prepared from mRNA extracted from yeast cells...

Based on its nature (aqueous solutions, physiological conditions, well-investigated labeling, and staining reactions) and the historical transition from slab-gel electrophoresis to CE, the main targets are biological and bioequivalent samples such as proteins, peptides, polynucleotides, oligonucleotides, and carbohydrates. 

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