title: Circular polymerase extension cloning for high-throughput cloning of complex and combinatorial DNA libraries.
abstract: High-throughput genomics, proteomics and synthetic biology studies require ever more efficient and economical strategies to clone complex DNA libraries or variants of biological modules. In this paper, we provide a protocol for a sequence-independent approach for cloning complex individual or combinatorial DNA libraries, and routine or high-throughput cloning of single or multiple DNA fragments. The strategy, called circular polymerase extension cloning (CPEC), is based on polymerase overlap extension and is therefore free of restriction digestion, ligation or single-stranded homologous recombination. CPEC is highly efficient, accurate and user friendly. Once the inserts and the linear vector have been prepared, the CPEC reaction can be completed in 10 min to 3 h, depending on the complexity of the gene libraries.
authors.affiliation: Department of Biomedical Engineering, Duke University, Durham, North Carolina, USA.
authors.forename: Jiayuan
authors.initials: J
authors.lastname: Quan
authors.forename: Jingdong
authors.initials: J
authors.lastname: Tian
mesh_terms: D003001:Cloning, Molecular / Q000379:methods*; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D005822:Genetic Vectors; D007763:Lac Operon / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D014170:Transformation, Genetic
chemical_list: D017931:DNA Primers