title: High-throughput cloning of human liver complete open reading frames using homologous recombination in Escherichia coli.
abstract: In this article, we describe a high-throughput cloning method, seamless enzyme-free cloning (SEFC), which allows one-step assembly of DNA fragments in vivo via homologous recombination in Escherichia coli. In the method, the desired open reading frame (ORF) is amplified by use of ORF-specific primers with flanking sequences identical to the two ends of a linearized vector. The polymerase chain reaction (PCR) product and the linearized vector are then cotransformed into E. coli cells, where the ORF is incorporated into the vector in vivo. SEFC is a simple, reliable, and inexpensive method of cloning in which PCR fragments are fused into expression vectors without unwanted amino acids or extra in vitro manipulations apart from the single PCR amplification step. Using this method, we successfully cloned human liver complete ORFs into the yeast AD and DB vectors and generated a clone resource of 4964 AD-ORFs and 4676 DB-ORFs in 3months. This approach will be useful for daily DNA cloning and for creating proteome-scale clone resources.
authors.affiliation: Institute of Biochemistry and Molecular Biology, Hubei University, Wuhan 430062, China.
authors.forename: Dewu
authors.initials: D
authors.lastname: Zhu
authors.forename: Xin
authors.initials: X
authors.lastname: Zhong
authors.forename: Rong
authors.initials: R
authors.lastname: Tan
authors.forename: Liang
authors.initials: L
authors.lastname: Chen
authors.forename: Guangrui
authors.initials: G
authors.lastname: Huang
authors.forename: Jing
authors.initials: J
authors.lastname: Li
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Sun
authors.forename: Li
authors.initials: L
authors.lastname: Xu
authors.forename: Jin
authors.initials: J
authors.lastname: Chen
authors.forename: Yangping
authors.initials: Y
authors.lastname: Ou
authors.forename: Ting
authors.initials: T
authors.lastname: Zhang
authors.forename: Dongxia
authors.initials: D
authors.lastname: Yuan
authors.forename: Zhen
authors.initials: Z
authors.lastname: Zhang
authors.forename: Wangyun
authors.initials: W
authors.lastname: Shu
authors.forename: Lixin
authors.initials: L
authors.lastname: Ma
mesh_terms: D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics*; D006801:Humans; D008099:Liver / Q000378:metabolism*; D016366:Open Reading Frames / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D040901:Proteomics / Q000379:methods*; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics