Biotin Labeling of DNA

HSV in human fibroblasts was detected using biotin-labeled HSV DNA probes, streptavidin-HRP complex, and enhanced CL substrate reagent for HRP [56], The presence of HSV DNA was observed in cells infected with clinical samples known to contain the HSV virus fixed at 48 h postinfection, with a sharp topographical localization and a good preservation of cellular morphology. 

A variation on the theme of conventional assay uses both lanthanide-labeled and biotin-labeled single strands to form split probes for sequence of target strands (Figure 12).120 When both of these bind to DNA, the complex binds (via the biotin residue) to a surface functionalized with streptavidin, immobilizing the europium and allowing assay to be carried out. This approach is already very sensitive to DNA sequence, since both sequences must match to permit immobilization of the lanthanide, but can be made even more sensitive by using PCR (the polymerase chain reaction) to enhance the concentration of DNA strands. In this way, initial concentrations corresponding to as few as four million molecules can be detected. This compares very favorably with radioimmunoassay detection limits. 

In a similar way, the use of oligonucleotide-immobilized beads enabled the realization of DNA sensitive biochips that could be used to detect biotin labelled sequence as 5.108 molecules59. 

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). 

The psoralen ring system can intercalate within double-stranded DNA or RNA and induce the formation of adducts with adjacent thymine bases (Figure 11.15). The furan-side and pyrone-side of the tricyclic rings in psoralen both can form cycloaddition products with the 5,6-double bond of thymine residues, which results in crosslinks between the DNA strands with a PEG-biotin label sticking out. 

Figure 27.1 Three common nucleoside triphosphate derivatives that can be incorporated into oligonucleotides by enzymatic means. The first two are biotin derivatives of pyrimidine and purine bases, respectively, that can be added to an existing DNA strand using either polymerase or terminal transferase enzymes. Modification of DNA with these nucleosides results in a probe detectable with labeled avidin or streptavidin conjugates. The third nucleoside triphosphate derivative contains an amine group that can be added to DNA using terminal transferase. The modified oligonucleotide then can be labeled with amine-reactive bioconjugation reagents to create a detectable probe.

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... 

When photobiotin is irradiated in the presence of DNA the reaction process nonselectively couples a biotin label to every 100-200 base residues. The result is an oligonucleotide probe detectable by the use of (strept)avidin conjugates. The uses of photobiotin for DNA or RNA modification are summarized in Chapter 11, Section 4. 

Assay for human immunodeficiency virus type 1 (HIV-1) proviral DNA in peripheral blood monuclear cells can be performed by PCR followed by detection of PCR products by electrochemiluminescence-labeled oligonucleotide probe [Tris-bipyridine ruthenium (II) complex]. Since one of the PCR primers is biotin-labeled at the 5 end, facile capture of the PCR product-probe complex can be accomplished on streptavidin-conjugated magnetic particles, prior to analysis in an electrochemiluminescence analyzer (S3). 

No phosphitylation of biotin nitrogen N2 was observed under these reaction conditions. The phosphoroamidite formed was designed for direct use in automated DNA synthesized using standard phosphoroamidite chemistry to introduce a photocleavable biotin label on the 5 -terminal phosphate of synthetic oligonucleosides. 

Several other methods are proposed, such as use of coulomb force between anionic DNA and cationic particle (7), use of biospecific affinity between biotin-labeled DNA and avidin-carrying particle (8), etc. Other methods for immobilization of DNA on the particle include reactions using cyanogen bromide, glutaraldehyde, and cyanuric chloride (9). 

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.

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