A Method for Generating User-defined Circular Single-stranded DNA from Plasmid DNA Using Golden Gate Intramolecular Ligation

Overview

Journal Biotechnol Bioeng

Publisher Wiley

Specialty Biochemistry

Date 2023 Jun 27

PMID 37366288

Authors

Isabell K Strawn

Paul J Steiner

Matilda S Newton

Zachary T Baumer

Timothy A Whitehead

Affiliations

Soon will be listed here.

Abstract

Construction of user-defined long circular single stranded DNA (cssDNA) and linear single stranded DNA (lssDNA) is important for various biotechnological applications. Many current methods for synthesis of these ssDNA molecules do not scale to multikilobase constructs. Here we present a robust methodology for generating user-defined cssDNA employing Golden Gate assembly, a nickase, and exonuclease degradation. Our technique is demonstrated for three plasmids with insert sizes ranging from 2.1 to 3.4 kb, requires no specialized equipment, and can be accomplished in 5 h with a yield of 33%-43% of the theoretical. To produce lssDNA, we evaluated different CRISPR-Cas9 cleavage conditions and reported a 52 ± 8% cleavage efficiency of cssDNA. Thus, our current method does not compete with existing protocols for lssDNA generation. Nevertheless, our protocol can make long, user-defined cssDNA readily available to biotechnology researchers.

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