This study describes the enzymatic synthesis of biotin-labeled nucleotides and their use as affinity probes for detecting and isolating specific DNA and RNA sequences. Biotin-labeled nucleotides were synthesized by covalently attaching biotin to the C-5 position of the pyrimidine ring via an allylamine linker. These nucleotides serve as efficient substrates for DNA and RNA polymerases. Biotin-labeled polynucleotides, whether single or double-stranded, are selectively retained on avidin-Sepharose even after extensive washing with high concentrations of urea, guanidine hydrochloride, or formamide. Additionally, they can be selectively immunoprecipitated using antibiotin antibodies and Staphylococcus aureus protein A. The biotin-avidin interaction has a very high binding affinity, making biotin-labeled nucleotides ideal for use in detection and isolation methods. These nucleotides can be incorporated into polynucleotides and used as probes in hybridization experiments. The study shows that biotin-labeled polynucleotides retain their hybridization characteristics even when substituted with a small number of biotin molecules. They are also resistant to enzymatic cleavage at certain restriction sites, which is important for their use in molecular biology applications. The synthesis of biotin-labeled nucleotides involved several steps, including the mercuration of UTP and dUTP, followed by alkylation with allylamine and subsequent coupling with biotin. The resulting biotin-labeled nucleotides were tested for their ability to function as substrates for various polymerases. Bio-dUTP was found to be an excellent substrate for E. coli DNA polymerase I, while Bio-UTP was a substrate for T7 RNA polymerase. These nucleotides were also shown to be selectively retained on avidin-Sepharose and immunoprecipitated using antibiotin antibodies. The study demonstrates that biotin-labeled nucleotides can be used as affinity probes for detecting and isolating specific DNA and RNA sequences. They are compatible with standard hybridization protocols and can be used in conjunction with immunological and affinity detection methods. The results suggest that biotin-labeled nucleotides can be used in a variety of molecular biology applications, including gene mapping and the detection of specific sequences in chromosomes, cells, and tissue sections.This study describes the enzymatic synthesis of biotin-labeled nucleotides and their use as affinity probes for detecting and isolating specific DNA and RNA sequences. Biotin-labeled nucleotides were synthesized by covalently attaching biotin to the C-5 position of the pyrimidine ring via an allylamine linker. These nucleotides serve as efficient substrates for DNA and RNA polymerases. Biotin-labeled polynucleotides, whether single or double-stranded, are selectively retained on avidin-Sepharose even after extensive washing with high concentrations of urea, guanidine hydrochloride, or formamide. Additionally, they can be selectively immunoprecipitated using antibiotin antibodies and Staphylococcus aureus protein A. The biotin-avidin interaction has a very high binding affinity, making biotin-labeled nucleotides ideal for use in detection and isolation methods. These nucleotides can be incorporated into polynucleotides and used as probes in hybridization experiments. The study shows that biotin-labeled polynucleotides retain their hybridization characteristics even when substituted with a small number of biotin molecules. They are also resistant to enzymatic cleavage at certain restriction sites, which is important for their use in molecular biology applications. The synthesis of biotin-labeled nucleotides involved several steps, including the mercuration of UTP and dUTP, followed by alkylation with allylamine and subsequent coupling with biotin. The resulting biotin-labeled nucleotides were tested for their ability to function as substrates for various polymerases. Bio-dUTP was found to be an excellent substrate for E. coli DNA polymerase I, while Bio-UTP was a substrate for T7 RNA polymerase. These nucleotides were also shown to be selectively retained on avidin-Sepharose and immunoprecipitated using antibiotin antibodies. The study demonstrates that biotin-labeled nucleotides can be used as affinity probes for detecting and isolating specific DNA and RNA sequences. They are compatible with standard hybridization protocols and can be used in conjunction with immunological and affinity detection methods. The results suggest that biotin-labeled nucleotides can be used in a variety of molecular biology applications, including gene mapping and the detection of specific sequences in chromosomes, cells, and tissue sections.