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Updated: Feb 11, 2026

Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins
Published on: February 25, 2017
In vitro synthesis of gene-length single-stranded DNA
Rémi Veneziano1, Tyson R Shepherd2, Sakul Ratanalert2,3
1Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA. rvenezia@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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