Current protocols in nucleic acid chemistry / edited by Serge L. Beaucage ... [et al.] 2013-03-01

Synthesis of threose nucleic acid (TNA) triphosphates and oligonucleotides by polymerase-mediated primer extension.

Su Zhang, Hanyang Yu, John C Chaput

Index: Curr. Protoc. Nucleic Acid Chem. Chapter 4 , Unit 4.54, (2013)

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Abstract

This unit describes the chemical synthesis of α-L-threofuranosyl nucleic acid (TNA) triphosphates for thymidine (T), guanosine (G), cytidine (C), and the diaminopurine (D) analog of adenosine and their incorporation into TNA oligonucleotides by enzyme-mediated polymerization of a DNA primer-template complex. Starting from suitably protected threofuranosyl nucleosides, TNA triphosphates are synthesized in a single-pot reaction and purified by ion-exchange and HPLC chromatography. Purified TNA triphosphates are diluted into stock solutions and used as substrates for the synthesis of TNA oligonucleotides. Oligonucleotide synthesis is accomplished using Therminator DNA polymerase, a commercial variant of the 9(o)N DNA polymerase bearing the A485L mutation.© 2013 by John Wiley & Sons, Inc.