DNA, biotinylated

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. in... 

Biotin-dUTP derivatives are formed by modification of the C-5 position of uridine. This location is not involved in hydrogen bonding activity with complementary DNA strands, thus hybridization efficiency is not immediately compromised. By contrast, biotin-dCTP or biotin-dATP derivatives involve modification of the bases at the N-4 position of cytosine and the N-6 position of adenine, locations directly involved in hydrogen bond formation with complementary bases. Thus, DNA biotinylation through the use of modified deoxynucleoside triphosphates to be incorporated into existing DNA strands may result in better activity of the probe if dUTP is used over dATP or dCTP. 

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). 

Fig. 26.2. Signals recorded for biotinylated SH-DNA, biotinylated DNA and background. C — 1 pM, ydmp = 5 iL, Timmob = 4°C, immoh = 12 h. Reprinted from Ref. [9], Copyright 2005, with permission from Elsevier.

FIG. 6 Successive coupling of two different biotinylated compounds with the DNA-STV conjugates 2 [33]. In a first step, a macromolecular functional component (FC, represented by the shaded ellipse), such as a biotinylated enzyme or oligonucleotide, is coupled. In a second step, a biotinylated low-molecular-weight modulator M, represented by the shaded sphere) is coupled to the remaining free biotin-binding sites. The modulator is used to modify the conjugate s hybridization properties or to supplement its functionality. 

FIG. 8 Synthesis of oligomeric DNA-STV conjugates 7 from 5, 5 -bis-biotinylated DNA and STV [49]. Note that the schematic structure of 7 is simplified, since a portion of the STV molecules function as tri- and tetravalent linker molecules between adjacent DNA fragments. 

Streptavidin-single-stranded DNA covalent conjugates were described as the building blocks for assembling nanostructured scaffolds [31], The amount and type of biotinylated ligands were used to modulate the affinity of duplex formation between solid-phase-bound nucleic acid templates and DNA-streptavidin conjugates. This system has been proposed for the design of fine-tuned sequence detection systems. 

A similar type of biotin-dendritic multimer also was used to boost sensitivity in DNA microarray detection by 100-fold over that obtainable using traditional avidin-biotin reagent systems (Stears, 2000 Striebel et al., 2004). With this system, a polyvalent biotin dendrimer is able to bind many labeled avidin or streptavidin molecules, which may carry enzymes or fluorescent probes for assay detection. In addition, if the biotinylated dendrimer and the streptavidin detection agent is added at the same time, then at the site of a captured analyte, the biotin-dendrimer conjugates can form huge multi-dendrimer complexes wherein avidin or streptavidin detection reagents bridge between more than one dendrimer. Thus, the use of multivalent biotin-dendrimers can become universal enhancers of DNA hybridization assays or immunoassay procedures. 

The reagent also has been used in a unique tRNA-mediated method of labeling proteins with biotin for nonradioactive detection of cell-free translation products (Kurzchalia et al., 1988), in creating one- and two-step noncompetitive avidin-biotin immunoassays (Vilja, 1991), for immobilizing streptavidin onto solid surfaces using biotinylated carriers with subsequent use in a protein avidin-biotin capture system (Suter and Butler, 1986), and for the detection of DNA on nitrocellulose blots (Leary et al., 1983). 

Sulfhydryl groups also can be added to 5 -phosphate end of DNA probes (Chapter 27, Section 2.2). Biotinylation at these sites avoids disruption of base pairing with complementary DNA targets, since the point of modification is restricted to a single end position on the oligonucleotide. 

Precipitate the sample to remove unreacted biotinylation reagent by adding 0.1 M potassium acetate and ethanol (1 2 ratio). Centrifuge and wash the biotinylated DNA pellet with ethanol, then dry it under nitrogen. The purified sample may be dissolved in water or buffer. 

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