2. Academic Validation
  3. De novo DNA synthesis using polymerase-nucleotide conjugates

De novo DNA synthesis using polymerase-nucleotide conjugates

  • Nat Biotechnol. 2018 Aug;36(7):645-650. doi: 10.1038/nbt.4173.
Sebastian Palluk 1 2 3,  Daniel H Arlow 1 2 4 5,  Tristan de Rond 1 2 6,  Sebastian Barthel 1 2 3,  Justine S Kang 1 2 7,  Rathin Bector 1 2 7,  Hratch M Baghdassarian 1 2 8,  Alisa N Truong 1 2,  Peter W Kim 1 9,  Anup K Singh 1 9,  Nathan J Hillson 1 2 10,  Jay D Keasling 1 2 5 7 8 11
Affiliations

Affiliations

  • 1 Joint BioEnergy Institute, Emeryville, California, USA.
  • 2 Biological Systems and Engineering Division, Lawrence Berkeley National Lab, Berkeley, California, USA.
  • 3 Department of Biology, Technische Universität Darmstadt, Darmstadt, Germany.
  • 4 Biophysics Graduate Group, UC Berkeley, Berkeley, California, USA.
  • 5 Institute for Quantitative Biosciences, UC Berkeley, Berkeley, California, USA.
  • 6 Department of Chemistry, UC Berkeley, Berkeley, California, USA.
  • 7 Department of Chemical and Biomolecular Engineering, UC Berkeley, Berkeley, California, USA.
  • 8 Department of Bioengineering UC Berkeley, Berkeley, California, USA.
  • 9 CBRN Defense and Energy Technologies, Sandia National Laboratories, Livermore, California, USA.
  • 10 DOE Joint Genome Institute, Walnut Creek, California, USA.
  • 11 Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmark.
PMID: 29912208 DOI: 10.1038/nbt.4173
Abstract

Oligonucleotides are almost exclusively synthesized using the nucleoside phosphoramidite method, even though it is limited to the direct synthesis of ∼200 mers and produces hazardous waste. Here, we describe an oligonucleotide synthesis strategy that uses the template-independent polymerase terminal deoxynucleotidyl transferase (TdT). Each TdT molecule is conjugated to a single deoxyribonucleoside triphosphate (dNTP) molecule that it can incorporate into a primer. After incorporation of the tethered dNTP, the 3' end of the primer remains covalently bound to TdT and is inaccessible to Other TdT-dNTP molecules. Cleaving the linkage between TdT and the incorporated nucleotide releases the primer and allows subsequent extension. We demonstrate that TdT-dNTP conjugates can quantitatively extend a primer by a single nucleotide in 10-20 s, and that the scheme can be iterated to write a defined sequence. This approach may form the basis of an enzymatic oligonucleotide synthesizer.

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