title: Quikgene: a gene synthesis method integrated with ligation-free cloning.
abstract: Gene synthesis is a convenient tool that is widely used to make genes for a variety of purposes. All current protocols essentially take inside-out approaches to assemble complete genes using DNA oligonucleotides or intermediate fragments. Here we present an efficient method that integrates gene synthesis and cloning into one step. Our method, which is evolved from QuikChange mutagenesis, can modify, extend, or even de novo synthesize relatively large genes. The genes are inserted directly into vectors without ligations or subcloning. We de novo synthesized a 600-bp gene through multiple steps of polymerase chain reaction (PCR) directly into a bacterial expression vector. This outside-in gene synthesis method is called Quikgene. Furthermore, we have defined an overlap region of a minimum of nine nucleotides in insertion primers that is sufficient enough to circularize PCR products for efficient transformation, allowing one to significantly reduce the lengths of primers. Taken together, our protocol greatly extends the current length limit for QuikChange insertion. More importantly, it combines gene synthesis and cloning into one step. It has potential applications for high-throughput structural genomics.
authors.affiliation: MOE Key Laboratory for Cell Biology, School of Life Sciences, Xiamen University, Fujian 361005, China.
authors.forename: Yanjun
authors.initials: Y
authors.lastname: Mao
authors.forename: Juanyu
authors.initials: J
authors.lastname: Lin
authors.forename: Aibin
authors.initials: A
authors.lastname: Zhou
authors.forename: Kunmei
authors.initials: K
authors.lastname: Ji
authors.forename: Jennifer S
authors.initials: JS
authors.lastname: Downey
authors.forename: Ruichuan
authors.initials: R
authors.lastname: Chen
authors.forename: Aidong
authors.initials: A
authors.lastname: Han
mesh_terms: D003001:Cloning, Molecular / Q000379:methods*; D017931:DNA Primers / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D016296:Mutagenesis; D016133:Polymerase Chain Reaction / Q000379:methods
chemical_list: D017931:DNA Primers