基于酿酒酵母克隆与错位热循环连接的环状全长人类线粒体DNA制备OA

[Objective]Mutations in human mitochondrial DNA(mtDNA)are associated with various diseases.However,the full-length sequence is unstable in prokaryotic hosts,and the in vitro preparation of large circular DNA fragments also remains challenging.Therefore,there is an urgent need for a new strategy to efficiently produce circularized full-length mtDNA,in order to overcome the critical technical bottleneck in mitochondrial genetic manipulation.[Method]First,to effectively overcome the host toxicity issues inherent in traditional prokaryotic cloning systems,we successfully cloned the full-length human mitochondrial genome into a yeast vector by leveraging the highly efficient homologous recombination system of Saccharomyces cerevisiae.Concurrently,site-directed editing was performed to eliminate the endogenous EcoR I restriction sites,thereby providing genetic screening markers for the artificially synthesized mtDNA.Subsequently,to address the challenge of circularizing linear PCR products,we developed a novel in vitro thermal cycling ligation strategy based on staggered linearized fragments.This method utilizes a thermostable ligase to drive the formation of heteroduplexe cohesive ends during denaturation-renaturation cycles,thereby achieving efficient circularization of long DNA fragments.[Result]Experimental results demonstrated the successful synthesis of the 16.6 kb full-length human mitochondrial genome.Restriction mapping and exonuclease resistance assays confirmed that the synthesized product consists of sequence-correct,nick-free,covalently closed circular DNA.[Conclusion]The yeast-assisted editing and in vitro cyclic assembly technological system established in this study not only overcomes the difficulty of preparing high-quality circular full-length mtDNA,but also provides a versatile genetic manipulation platform for constructing mitochondrial disease models,synthesizing artificial mitochondria,and developing mitochondrial gene therapy vectors.