In vitro synthesis of gene-length single-stranded DNA

Rémi Veneziano; Tyson R Shepherd; Sakul Ratanalert; Leila Bellou; Chaoqun Tao; Mark Bathe

doi:10.1038/s41598-018-24677-5

In vitro synthesis of gene-length single-stranded DNA

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

Authors

Rémi Veneziano¹, Tyson R Shepherd², Sakul Ratanalert^{2

3}, Leila Bellou², Chaoqun Tao², Mark Bathe⁴

Affiliations

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.

Publication types

Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

DNA, Single-Stranded / chemical synthesis*
Nucleotides / chemistry
Polymerase Chain Reaction / methods

Substances

DNA, Single-Stranded
Nucleotides