Proceedings of the National Academy of Sciences · 1981 · 1.1K citations · 21 references
Life SciencesBiochemistryMolecular BiologyEnzymologyMolecular BiotechnologyNucleic Acid BiochemistryAffinity ChromatographyNucleic Acid LabelingNucleic Acid PurificationNucleic Acid SynthesisOligonucleotide ChemistryPolymerase Chain ReactionAffinity-based ProbesEnzymatic Dna SynthesisEnzymatic SynthesisNucleic Acid Detection ProbesNucleic Acid Labeling ProbesNucleic Acid ProbesNucleotide AnalogsProbe Substrates
The authors aim to create biotin‑labeled polynucleotides that can serve as affinity probes for detecting and isolating specific DNA and RNA sequences. They synthesized dUTP and UTP analogs bearing a biotin moiety linked via an allylamine arm at the C‑5 position of the pyrimidine ring. The biotin‑labeled nucleotides proved efficient polymerase substrates, maintained normal hybridization behavior at low substitution levels, and bound avidin‑Sepharose and antibiotin antibody with high specificity, underscoring their utility as affinity probes.
Analogs of dUTP and UTP that contain a biotin molecule covalently bound to the C-5 position of the pyrimidine ring through an allylamine linker arm have been synthesized. These biotin-labeled nucleotides are efficient substrates for a variety of DNA and RNA polymerases in vitro. Polynucleotides containing low levels of biotin substitution (50 molecules or fewer per kilobase) have denaturation, reassociation, and hybridization characteristics similar to those of unsubstituted controls. Biotin-labeled polynucleotides, both single and double-stranded, are selectively and quantitatively retained on avidin-Sepharose, even after extensive washing with 8 M urea, 6 M guanidine hydrochloride, or 99% formamide. In addition, biotin-labeled polynucleotides can be selectively immunoprecipitated in the presence of antibiotin antibody and Staphylococcus aureus protein A. The unique features of biotin-labeled polynucleotides suggest that they will be useful affinity probes for the detection and isolation of specific DNA and RNA sequences.
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