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Sci Rep. 2018 Apr 25;8(1):6548. doi: 10.1038/s41598-018-24677-5.

In vitro synthesis of gene-length single-stranded DNA.

Author information

1
Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA. rvenezia@mit.edu.
2
Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA.
3
Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA.
4
Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA. mark.bathe@mit.edu.

Abstract

Single-stranded DNA (ssDNA) increases the likelihood of homology directed repair with reduced cellular toxicity. However, ssDNA synthesis strategies are limited by the maximum length attainable, ranging from a few hundred nucleotides for chemical synthesis to a few thousand nucleotides for enzymatic synthesis, as well as limited control over nucleotide composition. Here, we apply purely enzymatic synthesis to generate ssDNA greater than 15 kilobases (kb) using asymmetric PCR, and illustrate the incorporation of diverse modified nucleotides for therapeutic and theranostic applications.

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