Abstract
High-throughput genomics and the emerging field of synthetic biology demand ever more convenient, economical, and efficient technologies to assemble and clone genes, gene libraries and synthetic pathways. Here, we describe the development of a novel and extremely simple cloning method, circular polymerase extension cloning (CPEC). This method uses a single polymerase to assemble and clone multiple inserts with any vector in a one-step reaction in vitro. No restriction digestion, ligation, or single-stranded homologous recombination is required. In this study, we elucidate the CPEC reaction mechanism and demonstrate its usage in demanding synthetic biology applications such as one-step assembly and cloning of complex combinatorial libraries and multi-component pathways.
Highlights
Molecular cloning is a foundational technology for molecular biology and biotechnology
Sequence-independent cloning is largely based on homologous recombination and includes methods such as ligase-free [7] or ligation-independent cloning (LIC) [8], LIC with Uracil DNA glycosylase (UDG or USER cloning) [9,10], MAGIC [11], SLIC [12], In-Fusion (Clontech) [13], and PIPE [14]
We reasoned that it might be possible to eliminate this requirement by using the polymerase extension mechanism to extend doublestranded overlapping insert and vector to form a complete plasmid (Fig. 1A)
Summary
Molecular cloning is a foundational technology for molecular biology and biotechnology. Sequence-independent cloning is largely based on homologous recombination and includes methods such as ligase-free [7] or ligation-independent cloning (LIC) [8], LIC with Uracil DNA glycosylase (UDG or USER cloning) [9,10], MAGIC [11], SLIC [12], In-Fusion (Clontech) [13], and PIPE [14] These methods all have their own special characteristics and advantages, new developments especially the emergence of synthetic biology have put ever increasing demand for more accurate, efficient, convenient and economical cloning technologies for purposes such as creating complex combinatorial synthetic gene libraries, gene circuits and metabolic pathways
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