Biotin-dCTP

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. 

To label probes with haptens, both biotin-dNTPs (various suppliers biotin-dCTP from Gibco/Life Technologies is competitively priced and gives excellent results) and digoxigenin-dUTP (Boehringer Mannheim) have proved to be reliable. 

The pyrimidine nucleosides dUTP or dCTP can be modified at their C-5 position with a spacer arm containing a tag, such as a biotin group, and still remain good substrates for DNA polymerase. Enzymatic labeling with a biotin-modified pyrimidine nucleoside triphosphate is one of the most common methods of adding a detectable group to an existing DNA strand. 

Fig. 1. Labeling of degraded chromatin by the TUNEL assay. During apoptosis endogenous, endonucleases cleave chromatin in the hnker region between nucleosomes. The resulting nucleosome multimers are labeled by TdT and a dUTP analog with a detectable label (biotin, DIG, or FITC) shown as. The additional nucleotide in the reaction (here shown as dCTP) may be any dNTP and serves to extend the labeling reaction by preventing steric hindrance by two adjacent labeled dUTPs. (Abbreviations are as in text.)...

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). 

Labeling of probes for in situ hybridization relies on the incorporation of either a radioisotopic dNTP (e.g., dCTP), or of a nonisotopic molecule, such as biotin-7-dAlT or biotin-11-dUTP, by either nick translation or random priming. The site (s) of hybridization can then be seen using autoradiography with isotopic probes, or immunocytochemically if biotin is incorporated into the probe DNA. It is with the latter form of in situ hybridization methodology that this chapter is concerned. 

Biotin-14-dCTP catalog 19519-016 international distributors available on web site]... 

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