Synthesizing supercoiled circular DNA molecules in vitro.

ABSTRACT: Supercoiled (Sc) circular DNA, such as plasmids, are essential in molecular biology and hold strong therapeutic potential. However, they are typically produced in Escherichia coli, resulting in bacterial methylations, unnecessary sequences, and contaminants that hinder certain applications including clinical uses. These limitations could be avoided by synthesizing plasmids entirely in vitro, but synthesizing high-purity Sc circular DNA biochemically remains a significant technical challenge. To overcome this challenge, we have developed two novel biochemical methods for in vitro synthesis of Sc circular DNA. Linear DNA with two loxP sites in the same orientation is generated by polymerase chain reaction or rolling circle amplification. Cre recombinase efficiently converts the linear DNA in

SUBMITTER: Rezaei S 

PROVIDER: S-EPMC12421384 | biostudies-literature | 2025 Sep

REPOSITORIES: biostudies-literature