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Synthesize biotin-labeled dUTP and UTP analogs and evaluate their enzymatic incorporation into functional DNA and RNA affinity probes.

November 1, 1981Proceedings of the National Academy of SciencesOpen Access

Enzymatic synthesis of biotin-labeled polynucleotides: novel nucleic acid affinity probes.

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Synthesize biotin-labeled dUTP and UTP analogs and evaluate their enzymatic incorporation into functional DNA and RNA affinity probes.
Synthesized dUTP and UTP analogs covalently linked to biotin at the pyrimidine C-5 position via an allylamine linker arm.
Tested incorporation efficiency across multiple DNA and RNA polymerases in vitro.
Assessed probe hybridization kinetics, retention on avidin-Sepharose under harsh denaturants (8 M urea, 6 M guanidine hydrochloride, 99% formamide), and immunoprecipitation using anti-biotin antibodies.
Biotinylated nucleotides acted as efficient substrates for DNA and RNA polymerases, producing polynucleotides that maintained normal denaturation and hybridization behaviors at substitutions up to 50 molecules per kilobase.
Single- and double-stranded biotinylated polynucleotides demonstrated selective, quantitative retention on avidin-Sepharose despite rigorous washing with 8 M urea, 6 M guanidine hydrochloride, or 99% formamide.
Labeled polynucleotides were selectively immunoprecipitated using anti-biotin antibodies and Staphylococcus aureus protein A.

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Implication

In vitro experimental study demonstrates reliable enzymatic synthesis and avidin-binding affinity of biotinylated polynucleotides, suggesting utility for nucleic acid detection and isolation.

Cite This Study

Langer et al. (1981) studied this question.

synapsesocial.com/papers/6a0cc7259d761985b14a4835 https://doi.org/10.1073/pnas.78.11.6633

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Implication

In vitro experimental study demonstrates reliable enzymatic synthesis and avidin-binding affinity of biotinylated polynucleotides, suggesting utility for nucleic acid detection and isolation.

Enzymatic synthesis of biotin-labeled polynucleotides: novel nucleic acid affinity probes.

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Discussion

Implication

Cite This Study

Enzymatic synthesis of biotin-labeled polynucleotides: novel nucleic acid affinity probes.

Key Points

Discussion

Implication

Cite This Study

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