Terminal Deoxynucleotidyl Transferase
Overview
Terminal Deoxynucleotidyl Transferase (TdT) is a template-independent DNA polymerase, meaning that it can add DNA bases to an existing single strand of DNA without the sequence being dictated by a longer complementary strand. TdT must have a starting strand of DNA 3 base pairs long, and it adds to the 3' end of the strand like most polymerases. TdT can use many different nucleotides as substrates, incorporating a huge variety of natural and synthetic nucleotides, whether blocked or unblocked, although it prefers to add dGTP and dCTP and avoids dATP. It is also suspected that TdT adds completely distributively, detaching from and reattaching to each strand between addition events instead of staying on one strand of DNA. In nature, TdT is used for DNA repair and to generate random antibodies in the immune system. SSI Biology is using recombinant bovine TdT to extend starting strands of DNA with an arbitrary sequence of bases for de novo DNA synthesis.
Structure
TdT is shaped like a right hand like most polymerases, with a metal ion-containing "palm" domain where the extension reaction occurs, "fingers" to align the DNA strand and nucleotide properly, and an "index finger" and "thumb" to guide nucleotides into the enzyme active site. However, TdT contains a 16-amino acid loop that only lets in single stranded DNA for binding, and not double stranded DNA.
Divalent Cations
TdT uses divalent cations as catalysts to extend DNA, and the efficiency of the reaction is affected by the type of cation used. Zinc and magnesium cations work best for extension, with magnesium causing TdT to prefer dGTP and dATP. Most commercial TdT buffers (such as the one SSI Biology currently uses) contain cobalt, which leads to preferential addition of dCTP and dTTP. Ethylenediaminetetraacetic acid (EDTA) inactivates TdT by chelating all of the divalent cations so that the addition reaction cannot occur.
Generic TdT Extension Protocol
TdT is incubated with a starting primer, nucleotides, TdT buffer, and nuclease-free water at 37C for thirty minutes to an hour. Once the reaction is complete, TdT can be heat-inactivated by raising temperature to 70C for 10 minutes. EDTA can also be added to inactivate the enzyme and stop the reaction. TdT should be stored at -20C.
References
Motea, Edward A., and Anthony J. Berdis. “Terminal Deoxynucleotidyl Transferase: The Story of a Misguided DNA Polymerase.” Biochimica et biophysica acta 1804.5 (2010): 1151–1166. PMC. Web. 10 Sept. 2017.