Current and Emerging Methods for the Synthesis of Single-Stranded DNA

Abstract

Methods for synthesizing arbitrary single-strand DNA (ssDNA) fragments are rapidly becoming fundamental tools for gene editing, DNA origami, DNA storage, and other applications. To meet the rising application requirements, numerous methods have been developed to produce ssDNA. Some approaches allow the synthesis of freely chosen user-defined ssDNA sequences to overcome the restrictions and limitations of different length, purity, and yield. In this perspective, we provide an overview of the representative ssDNA production strategies and their most significant challenges to enable the readers to make informed choices of synthesis methods and enhance the availability of increasingly inexpensive synthetic ssDNA. We also aim to stimulate a broader interest in the continued development of efficient ssDNA synthesis techniques and improve their applications in future research.

On‐Surface Synthesis of Polyferrocenylene and its Single‐Chain Conformational and Electrical Transport Properties

“…In this sense, the polymerization of monomers is perfectly mastered only by enzymatic machineries for the production of defined nucleic acids, proteins, or polysaccharides. Automated solid-phase synthesis, which has undergone remarkable development over time, can also deliver long sequences of polynucleotides (routinely around 200-mers), [78,79] polypeptides (up to 164-mer, [80] without native chemical ligation), [81] or polysaccharides (up to multiple-branched 151-mer). [82] However, such a methodology would not be easily adaptable for the preparation of polyferrocenylene 2 as the problem of its insolubility would persist.…”

Synthesis of DNA Origami Scaffolds: Current and Emerging Strategies

“…Alternative ssDNA synthesis methods continue to be developed and show promise toward potentially synthesizing full-length DNA origami scaffolds [ 115 ]. These emerging methods include nicking strand displacement amplification (nSDA), primer exchange reaction (PER), and terminal deoxynucleotidyl transferase-based (TdT) synthesis.…”

Advances in Genome Editing and Application to the Generation of Genetically Modified Rat Models

“…This particularly interesting approach will be applied eventually to generate genetically modified rat models. Cas9 protein allowing rapid and more efficient editing (Kim et al, 2014;Ménoret et al, 2015) Large editing toolbox variants (Table 3) Improved chromatin accessibility (Chen F. et al, 2017;Ding et al, 2019) Cas9 engineered to activate repair pathways (Charpentier et al, 2018;Tran et al, 2019) Cas9 engineering to be degraded in G1 (Gutschner et al, 2016;Charpentier et al, 2018;Lomova et al, 2019) Filippova et al, 2019) Essential sequence, secondary structures and functional modules of gRNA (Briner et al, 2014;Kartje et al, 2018) Overlapping gRNA (Jang et al, 2018) gRNA engineering to activate repair pathways (Nakade et al, 2018;Tran et al, 2019) IVT sgRNA Easy to produce and use (Hao et al, 2020) Chemical modification (Renaud et al, 2016; Insertion close to cut site (Inui et al, 2014; 3 overhang DNA donor Hirotsune et al, 2020) Carry to cut site by Cas9 (Ma et al, 2017;Aird et al, 2018;Gu et al, 2018;Ling et al, 2020; Carry to cut site by gRNA (Carlson-Stevermer et al, 2017;Lee et al, 2017) Carry to cut site by DNA donor engineering (Nguyen et al, 2020) DNA donor in vivo excision from plasmid (Aida et al, 2016;Yao et al, 2017;Zhang et al, 2017) lsDNA…”