title: Rapid assembly of multiple DNA fragments through direct transformation of PCR products into E. coli and Lactobacillus.
abstract: This article describes a rapid, highly efficient and versatile method for seamlessly assembling multiple DNA fragments into a vector at any desired position. The inserted fragments and vector backbone were amplified by high-fidelity PCR containing 20 bp to 50 bp overlapping regions at 3' and/or 5' termini. These linearised fragments were equimolarly mixed, and then cyclised in a prolonged overlap extension PCR without adding primers. The resulting PCR products were DNA multimers that could be directly transformed into host strains, yielding the desired chimeric plasmid. The proposed method was illustrated by constructing an Escherichia coli co-expression vector. The feasibility of the method in Lactobacillus was further validated by assembling an E. coli-Lactobacillus shuttle vector. Results showed that three to four fragments could be simultaneously and precisely inserted in a vector in only 2-3 days using the proposed method. The acceptable transformation efficiency was determined through the tested host strains; more than 95% of the colonies were positive transformants. Therefore, the proposed method is sufficiently competent for high-efficiency insertion of multiple DNA fragments into a plasmid and has theoretically good application potential for gene cloning and protein expression because it is simple, easy to implement, flexible and yields highly positive clones.
authors.affiliation: College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China; College of Animal Science and Technology, Henan University of Science and Technology, Luoyang 471003, China.
authors.forename: Pinghua
authors.initials: P
authors.lastname: Cao
authors.affiliation: College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Wang
authors.affiliation: College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China.
authors.forename: Guangxian
authors.initials: G
authors.lastname: Zhou
authors.affiliation: College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China.
authors.forename: Yaoyue
authors.initials: Y
authors.lastname: Wang
authors.affiliation: College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China. Electronic address: chenyulin@nwsuaf.edu.cn.
authors.forename: Yulin
authors.initials: Y
authors.lastname: Chen
mesh_terms: D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D007778:Lactobacillus / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D014170:Transformation, Genetic
chemical_list: D004247:DNA
keywords: Multiple fragment assembly; Prolong overlap extension PCR; Seamless cloning