Synthesis and hybridization of a series of biotinylated oligonucleotides

Abstract

A series of oligonucleotides containing biotin-11-dUMP at various positions were synthesized and compared in quantitative, colorimetric hybridization-detection studies. A deoxyuridine phosphoramidite containing a protected allylamino sidearm was synthesized and used in standard, automated synthesis cycles to prepare oligonucleotides with allylamino residues at various positions within a standard 17-base sequence. Biotin substituents were subsequently attached to the allylamino sidearms by reaction with N-biotinyl-6-aminocaproic acid N-hydroxysuccinimide ester. These oligomers were hybridized to target DNA immobilized on microtiter wells (ELISA plates), and were detected with a streptavidin-biotinylated horseradish peroxidase complex using hydrogen peroxide as substrate and o-phenylenediamine as chromogen. We found that the sensitivity of detection of target DNA by biotin-labeled oligonucleotide probes was strongly dependent upon the position of the biotin label. Oligonucleotides containing biotin labels near or off the ends of the hybridizing sequence were more effective probes than oligonucleotides containing internal biotin labels. An additive effect of increasing numbers of biotin-dUMP residues was found for some labeling configurations.

Evaluation of the Performance of Two Carbodiimide-Based Cyanine Dyes for Detecting Changes in mRNA Expression with DNA Microarrays

“…Similarly, a previous study revealed that internal biotin labels attached via aminoallyl-dNTP subtly affect hybridization thermodynamics and duplex stability, and end-labeling of the DNA target enhances hybridization intensity (22). Nevertheless, we have shown that the carbodiimide-based cyanine dyes produce consistent data when repeatedly applied to analyses under the same conditions.…”

Rotavirus detection by dot blot hybridization assay using a non-radioactive synthetic oligodeoxynucleotide probe

“…The probe used in this work was labelled with biotin-7-dATP by tailing at the 3' end with terminal deoxynucleotydyl transferase [33]. We have selected this system because it allows the incorporation of an adequate number of biotin residues.…”

“…We have selected this system because it allows the incorporation of an adequate number of biotin residues. It has been shown that oligonucleotide probes with unhybridized 'tails' of biotin-dNTP residues provide a non-radioactive detection substrate that is more sensitive than one with biotin incorporated into internucleotide linkage by chemical synthetic methods [33].…”

Nucleic acid analysis by sandwich hybridization

“…01-igonucleotides can be labelled with radioisotopes (85,86), or with nonradioactive marker groups (87)(88)(89)(90) action can be improved by increasing the concentration of probe, without the danger of probe re-association, and removal from the hybridization equilibrium. The main disadvantages of oligonucleotides are, firstly, that their labelling results in probes with specific activities 10-100-fold less than those produced by random hexanucleotide priming (33,34) or nick translation (32) and, secondly, that a knowledge of the nucleotide sequence of the target nucleic acid is a prerequisite to oligonucleotide synthesis.…”