Terminal deoxynucleotidyl transferase (TdT), is a template-independent DNA polymerase that catalyzes the incorporation of deoxynucleotides at the 3'-hydroxyl terminus of DNA, accompanied by the release of inorganic phosphate. TdT does not require a template and will not copy one. Reaction conditions and some applications are described in this unit, including cloning DNA fragments, labeling the 3' terminus of DNA with (32)P or nonradioactive tags, synthesizing model polydeoxynucleotide homopolymers, and detecting DNA damage and apoptotic cells.
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A route to prepare ribonucleoside triphosphates featuring a 3'-aminoxy (3'-O-NH ) removable blocking group is reported here. We then show that versions of two DNA polymerases, human DNA polymerase theta (Polθ) and mimiviral PrimPol, accept these triphosphates as substrates to add single nucleotides to an RNA primer under engineered conditions. Cleaving the O-N bond in the 3'-O-NH group within the extended primer regenerates the 3'-OH group, facilitating subsequent polymerase cycles that add a second, selected, nucleotide.
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Department of Biomedical Engineering, University of California, Irvine, California 92697, United States.
Terminal deoxynucleotidyl transferase (TdT) is a unique DNA polymerase capable of template-independent extension of DNA. TdT's DNA synthesis ability has found utility in DNA recording, DNA data storage, oligonucleotide synthesis, and nucleic acid labeling, but TdT's intrinsic nucleotide biases limit its versatility in such applications. Here, we describe a multiplexed assay for profiling and engineering the bias and overall activity of TdT variants with high throughput.
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MOE International Joint Research Laboratory on Synthetic Biology and Medicines, School of Biology and Biological Engineering, South China University of Technology, 510006 Guangzhou, China.
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