De novo DNA synthesis using polymerasenucleotide conjugates

Research output: Contribution to journalJournal article – Annual report year: 2018Researchpeer-review

DOI

  • Author: Palluk, Sebastian

    Joint Bioenergy Institute, United States

  • Author: Arlow, Daniel H

    University of California at Berkeley, United States

  • Author: de Rond, Tristan

    Joint Bioenergy Institute, United States

  • Author: Barthel, Sebastian

    Technische Universität Darmstadt, Germany

  • Author: Kang, Justine S

    University of California at Berkeley, United States

  • Author: Bector, Rathin

    University of California at Berkeley, United States

  • Author: Baghdassarian, Hratch M

    University of California at Berkeley, United States

  • Author: Truong, Alisa N

    Joint Bioenergy Institute, United States

  • Author: Kim, Peter W

    Sandia National Laboratories, United States

  • Author: Singh, Anup K.

    Sandia National Laboratories, United States

  • Author: Hillson, Nathan J.

    Joint Genome Institute, United States

  • Author: Keasling, Jay D

    Synthetic Biology Tools for Yeast, Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kogle Allé 6, 2970, Hørsholm, Denmark

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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.
Original languageEnglish
JournalNature Biotechnology
Volume36
Issue number7
Pages (from-to)645-650
ISSN1087-0156
DOIs
Publication statusPublished - 2018
CitationsWeb of Science® Times Cited: No match on DOI

ID: 151637529