De novo DNA synthesis using polymerase-nucleotide conjugates.
Identifieur interne : 000A14 ( Main/Exploration ); précédent : 000A13; suivant : 000A15De novo DNA synthesis using polymerase-nucleotide conjugates.
Auteurs : Sebastian Palluk [États-Unis] ; Daniel H. Arlow [États-Unis] ; Tristan De Rond [États-Unis] ; Sebastian Barthel [États-Unis] ; Justine S. Kang [États-Unis] ; Rathin Bector [États-Unis] ; Hratch M. Baghdassarian [États-Unis] ; Alisa N. Truong [États-Unis] ; Peter W. Kim [États-Unis] ; Anup K. Singh [États-Unis] ; Nathan J. Hillson [États-Unis] ; Jay D. Keasling [États-Unis]Source :
- Nature biotechnology [ 1546-1696 ] ; 2018.
Descripteurs français
- KwdFr :
- Composés organiques du phosphore (), DNA nucleotidylexotransferase (), DNA nucleotidylexotransferase (génétique), DNA-directed DNA polymerase (), DNA-directed DNA polymerase (génétique), Nucléosides (), Nucléosides (génétique), Oligonucléotides (), Oligonucléotides (biosynthèse), Oligonucléotides (génétique), Réplication de l'ADN (génétique).
- MESH :
English descriptors
- KwdEn :
- DNA Nucleotidylexotransferase (chemistry), DNA Nucleotidylexotransferase (genetics), DNA Replication (genetics), DNA-Directed DNA Polymerase (chemistry), DNA-Directed DNA Polymerase (genetics), Nucleosides (chemistry), Nucleosides (genetics), Oligonucleotides (biosynthesis), Oligonucleotides (chemistry), Oligonucleotides (genetics), Organophosphorus Compounds (chemistry).
- MESH :
- chemical , biosynthesis : Oligonucleotides.
- chemical , chemistry : DNA Nucleotidylexotransferase, DNA-Directed DNA Polymerase, Nucleosides, Oligonucleotides, Organophosphorus Compounds.
- chemical , genetics : DNA Nucleotidylexotransferase, DNA-Directed DNA Polymerase, Nucleosides, Oligonucleotides.
- genetics : DNA Replication.
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.
DOI: 10.1038/nbt.4173
PubMed: 29912208
Affiliations:
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Le document en format XML
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<term>DNA-Directed DNA Polymerase (genetics)</term>
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<front><div type="abstract" xml:lang="en">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.</div>
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