Biotin-dATP

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. 

Other nucleotides, such as dCTP, can be substituted for dATP at equal concentrations. The addition of unlabeled dNTPs to the reaction allows for a longer tail to be added by TdT without the problem of steric hindrance caused by the modification on the nucleotide (biotin, DIG, or FITC). Fluorescein-labeled nucleotides should be protected from light at all times to avoid loss of signal. 

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). 

Nick translation kit Biotin-7-dATP or Biotin-11-dUTP. 

Label 1 pg of DNA probe with biotin-11-dUTP or biotin-7-dATP by nick... 

Biotin-7-dATP (Gibco) or biotin-ll-dUTP (Enzo, NY) (seeNote 1). 

Biotin-l 1-dUTP and biotin-7-dATP have both been used successfully to label probe and genomic DNA. Biotin-l 1-dUTP is now only available direct from Enzo Diagnostics (NY). 

Biotin-1 l-deoxyuridine-5 -triphosphate (BiodUTP) and reagents for its incorporation into DNA by nick translation are obtained commercially. The products from Bethesda Research Laboratories (BRL, Gaithersbui, MD) are acceptable. BRL now provides a prepackaged kit (BioNick) containing necessary supplies and employing biotin-14-dATP as the source of biotin. The concentration of the resulting biotinylated DNA is determined by the histochemical method for biotin (below). Adequate instructions are provided with these kits. 

N -(6-Aminohexyl)dATP (AHdATP), which is virtually as efficiently incorporated as dATP, via its primary amine group on the carbon chain, provides a reactive group for an N-hydroxysuccinimide (NHS) ester of any hapten (including biotin, various haptens for which antibodies are available, fluorescent dyes, etc.). Although this procedure has been described for nick translation (Gebeyehu et al., 1987), it is more useful in random priming. In this procedure, the... 

Optimize probes label probe is preferably an RNA probe or a is DNA probe of at least 100 bases ( > 50% GC) with or biotin labels whereas the binder probe should be an oligomer of 40 bases (> 50% GC) with a dA-tail length of 100-200 bases (tailed overnight with 1000 units of TdTase, 0.01 mM oligomer and 2 mM dATP as described in Section 7.7.2). 

Examples shown here have been previously published (Henrich, 1990 Karger et al., 1990). This procedure has also been used to introduce ions, nucleotides and analogs (ATP, biotin-14-dATP), fluorescein-labeled peptides, and fluorophores. 

Other nucleotides The incorporation of a dATP analog, 2-aminopurine-dNTP (dAPTP), is decreased 3-10 times in the presence of an equal concentration of dATP (40). Although inefficient, Pol I can also incorporate various other nucleotide analogs such as dITP, 7-deaza-dGTP, biotin- or digoxigenin-labeled dUTP, and fluorescein-12-dUTP. 

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