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Nat Biotechnol. 2018 Aug;36(7):645-650. doi: 10.1038/nbt.4173. Epub 2018 Jun 18.

De novo DNA synthesis using polymerase-nucleotide conjugates.

Palluk S1,2,3, Arlow DH1,2,4,5, de Rond T1,2,6, Barthel S1,2,3, Kang JS1,2,7, Bector R1,2,7, Baghdassarian HM1,2,8, Truong AN1,2, Kim PW1,9, Singh AK1,9, Hillson NJ1,2,10, Keasling JD1,2,5,7,8,11.

Author information

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.

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.

PMID:
29912208
DOI:
10.1038/nbt.4173
[Indexed for MEDLINE]
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