High-throughput cloning of human liver complete open reading frames using homologous recombination in Escherichia coli.
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.
Zhu
Dewu
D
Institute of Biochemistry and Molecular Biology, Hubei University, Wuhan 430062, China.
Zhong
Xin
X
Tan
Rong
R
Chen
Liang
L
Huang
Guangrui
G
Li
Jing
J
Sun
Xiaoyan
X
Xu
Li
L
Chen
Jin
J
Ou
Yangping
Y
Zhang
Ting
T
Yuan
Dongxia
D
Zhang
Zhen
Z
Shu
Wangyun
W
Ma
Lixin
L
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