The authors have synthesized biotin-labeled nucleotides (Bio-UTP and Bio-dUTP) that serve as efficient substrates for various DNA and RNA polymerases in vitro. These biotin-labeled polynucleotides exhibit similar denaturation, reassociation, and hybridization characteristics to their unsubstituted counterparts. They are selectively retained on avidin-Sepharose and can be immunoprecipitated using antibiotin antibodies and Staphylococcus aureus protein A. The unique properties of these biotin-labeled polynucleotides suggest their potential as useful affinity probes for detecting and isolating specific DNA and RNA sequences. The study demonstrates that a limited number of biotin molecules (50 or fewer per kilobase) is sufficient for effective retention and immunoprecipitation, making them suitable for standard hybridization protocols. The authors also discuss the implications of these findings for gene mapping, immunoprecipitation techniques, and enrichment of specific gene sequences.The authors have synthesized biotin-labeled nucleotides (Bio-UTP and Bio-dUTP) that serve as efficient substrates for various DNA and RNA polymerases in vitro. These biotin-labeled polynucleotides exhibit similar denaturation, reassociation, and hybridization characteristics to their unsubstituted counterparts. They are selectively retained on avidin-Sepharose and can be immunoprecipitated using antibiotin antibodies and Staphylococcus aureus protein A. The unique properties of these biotin-labeled polynucleotides suggest their potential as useful affinity probes for detecting and isolating specific DNA and RNA sequences. The study demonstrates that a limited number of biotin molecules (50 or fewer per kilobase) is sufficient for effective retention and immunoprecipitation, making them suitable for standard hybridization protocols. The authors also discuss the implications of these findings for gene mapping, immunoprecipitation techniques, and enrichment of specific gene sequences.