Journal
NATURE BIOTECHNOLOGY
Volume 36, Issue 7, Pages 645-+Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/nbt.4173
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Funding
- DOE Joint BioEnergy Institute by the US Department of Energy, Office of Science, Office of Biological and Environmental Research [DE-AC02-05CH11231]
- Synthetic Biology Engineering Research Center (SynBERC) through National Science Foundation [NSF EEC 0540879]
- NIH through NIGMS [GM-08295]
- ERASynBio [81861: SynPath]
- DOE Joint Genome Institute by the US Department of Energy, Office of Science, Office of Biological and Environmental Research [DE-AC02-05CH11231]
- US Department of Energy's National Nuclear Security Administration [DE-NA0003525]
- United States Government
- Department of Energy
- DOE Public Access Plan
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Oligonucleotides are almost exclusively synthesized using the nucleoside phosphoramidite method, even though it is limited to the direct synthesis of similar to 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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