circular polymerase extension cloning of complex gene libraries and pathways圆形的聚合酶扩展复杂基因的克隆库和途径.pdfVIP

Circular Polymerase Extension Cloning of Complex Gene Libraries and Pathways Jiayuan Quan, Jingdong Tian* Department of Biomedical Engineering Institute for Genome Sciences and Policy, Duke University, Durham, North Carolina, United States of America 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. Citation: Quan J, Tian J (2009) Circular Polymerase Extension Cloning of Complex Gene Libraries and Pathways. PLoS ONE 4(7): e6441. doi:10.1371/ journal.pone.0006441 Editor: Paulo Lee Ho, Instituto Butantan, Brazil Received May 10, 2009; Accepted June 29, 2009; Published July 30, 2009 Copyright: 2009 Quan, Tian. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded in part by the Arnold and Mablel Beckman Foundation () and by the Hartwell Foundation (). J.Q was initially supported by the Duke University BME graduate program. The funders had no role in study design, data colle