Oligonucleotide amine-modified

Call et al. (2001) of the Pacific Northwest National Laboratory in Richland, WA, also studied the immobilization of unmodified oligonucleotides. Amine-modified and unmodified oligonucleotides could be attached to epoxysilane slides (covalent attachment) or acid-washed slides (noncovalent attachment) under the same conditions by printing in an alkaline-sodium... 

Smith and co-workers (194) have used this chemistry to prepare carboxyl-modified Si(lll) surfaces at which polylysine-tethered DNA is electrostatically adsorbed (Fig. 60). An alternative approach involved covalent attachment of a pre-synthesized oligonucleotide bearing a terminal carboxyl group to an amine-modified Si(001) surface (195). 

SIAB and sulfo-SIAB have been used to make a high-capacity RNA affinity column for the purification of human IRP1 and IRP2 (Allerson et al., 2003), to couple antibodies or Fab fragments to amine-modified microparticles (Harma et al., 2000), and in the attachment of oligonucleotides to surfaces for detection arrays (Adessi et al., 2000). 

Dissolve the amine-modified oligonucleotide to be thiolated in 250pi of 50mM sodium phosphate, pH 7.5. 

Prepare an amine-modified oligonucleotide according to any of the protocols discussed in Section 2.1 (this chapter). Dissolve or buffer-exchange the oligo into 0.1M sodium borate, 2mM EDTA, pH 8.25, at a concentration of 9 nmol (2.0 A2gonm units) in 15 pi. 

The following protocol is a generalized method for labeling amine-modified oligonucleotides with a fluorescent probe, such as FITC. It is based on the method of Morrison (1992). 

Perhaps the most common method of DNA biotinylation is through enzymatic incorporation with the use of a biotin-labeled deoxynucleoside triphosphate. First reported by Langer et al. and Leary et al. in 1981, the procedure is probably the most popular nonradioactive labeling technique reported for oligonucleotide probes. Although biotinylated derivatives of dCTP and dATP are reported in the literature, by far the most frequently employed derivative is biotin—dUTP prepared from the reaction of an amine-modified dUTP with an amine-reactive biotinylation reagent, such as NHS-LC—biotin (Chapter 8, Section 3.1). 

Prepare an amine-modified oligonucleotide using any of the methods of Sections 2.1 or 1. Dissolve or buffer exchange 70 pg of the oligo into 25 pi of 0.1 M sodium borate, pH 9.3. 

Using planar waveguides made out of Hydex glass, one study demonstrated detection of 500 nM bulk concentration of 24-base target oligonucleotides [30], The surface of the resonator in this case is treated with an epoxysilane compound that reacted with amine-modified probe nucleotides. However, this particular study includes no characterization of limits of detection. 

The PCR template can either be prepared plasmid DNA or bacterial cell lysates. The primers can be standard oligonucleotides or 5-amine modified, depending on what slide binding chemistry is adopted (see Subheading 5.1.2.). 

Carbodi-imides are used to mediate the formation of amide linkage betwen a carboxylate and an amine or phosphoramidate linkages between a phosphate and an amine [12]. The following is essentially the method of Rockwood [13] and is modified to give a phospho-diester link between the terminal monophosphate of the oligonucleotide and the hydroxyl group of 2-hydroxyethyl disulfide (HEDS) [14]. 

The syntheses, photophysical and electrochemical properties of [Ir(ppy)2(phen-NS-5)]PF6, (181), [Ir(ppy)2(phen-NHCOCH2I-5)]PF6, (182), and [Ir(ppy)2(phen-NH2-5)]PF6 are reported.342 Complexes (181) and (182) have been used to label amine- and sulfhydryl-modified oligonucleotides and human serum albumin to give luminescent bioconjugates. 

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