title: Robust one-tube Ω-PCR strategy accelerates precise sequence modification of plasmids for functional genomics.
abstract: Functional genomics requires vector construction for protein expression and functional characterization of target genes; therefore, a simple, flexible and low-cost molecular manipulation strategy will be highly advantageous for genomics approaches. Here, we describe a Ω-PCR strategy that enables multiple types of sequence modification, including precise insertion, deletion and substitution, in any position of a circular plasmid. Ω-PCR is based on an overlap extension site-directed mutagenesis technique, and is named for its characteristic Ω-shaped secondary structure during PCR. Ω-PCR can be performed either in two steps, or in one tube in combination with exonuclease I treatment. These strategies have wide applications for protein engineering, gene function analysis and in vitro gene splicing.
authors: State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, College of Life Sciences, South China Agricultural University, Guangzhou 510642, China. lotichen@scau.edu.cn
authors: Letian
authors: L
authors: Chen
authors: Fengpin
authors: F
authors: Wang
authors: Xiaoyu
authors: X
authors: Wang
authors: Yao-Guang
authors: YG
authors: Liu
mesh_terms: D003001:Cloning, Molecular; D023281:Genomics / Q000379:methods*; D010957:Plasmids / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*