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Free-running enzymatic oligonucleotide synthesis for data storage applications

biorxiv. 2018; 
M.A. Jensen, P.B. Griffin, R. W. Davis
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PCR Cloning and Subcloning For amplification of ACTC-TdT polymerized reaction product for Sanger sequencing: FRS reaction, 5 ul, Taq polymerase (NEB) 0.5 ul, Taq buffer (1x), dNTPs (NEB) final 0.2 mM, 5 pm forward primer (FRSi initiator), 5 pm reverse primer (T40), water added to 25 ul. Thermal-cycling conditions using an ABI Veriti: 95 °C/30 40* [95 °C/15s | 45 °C/30s | 68 °C/30s] 68 °C/3m. For sequence analysis, we poly-adenylated the 3’ end of each sample to introduce an anchor for T40 to anneal in PCR amplification; the initiator sequence (CTGAGTTCGCTACAGTGTACTC) was also used as a forward primer (Tm values, 50 °C and 55 °C, respectively). We followed the manufacturer’s recommended protocol (Invitrogen, TOPO TA Cloning Kit for Sequencing, 450030). Sequencing was performed by GenScript; for results in Fig. 1, PCR product was used; for results in Fig. 3, target samples were inserted into the pCR-4 TOPO vector, and plasmid purified. Get A Quote

摘要

Here we present preliminary results for a method of oligodeoxynucleotide synthesis using terminal deoxynucleotidyl transferase (TdT) to generate mixed base homopolymer runs of defined sequence. In a process we have termed Free-Running Synthesis (FRS), we allow TdT to freely add multiple bases of a given monomer. With this method, homopolymeric runs can be used for writing DNA in a data storage capacity, where a stretch of A’s, for example, will be read as a single base, and the transitions between successive homopolymer runs encodes information. As a proof-of-concept, we demonstrate homopolymer additions of A, C and T onto the 3’ end of a 22 base initiator strand.

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