title: The MASTER (methylation-assisted tailorable ends rational) ligation method for seamless DNA assembly.
abstract: Techniques for assembly of designed DNA sequences are important for synthetic biology. So far, a few methods have been developed towards high-throughput seamless DNA assembly in vitro, including both the homologous sequences-based system and the type IIS-mediated system. Here, we describe a novel method designated 'MASTER Ligation', by which multiple DNA sequences can be seamlessly assembled through a simple and sequence-independent hierarchical procedure. The key restriction endonuclease used, MspJI, shares both type IIM and type IIS properties; thus, it only recognizes the methylation-specific 4-bp sites, (m)CNNR (R = A or G), and cuts DNA outside of the recognition sequences. This method was tested via successful assembly of either multiple polymerase chain reaction amplicons or restriction fragments of the actinorhodin biosynthetic cluster of Streptomyces coelicolor (∼29 kb), which was further heterologously expressed in a fast-growing and moderately thermophilic strain, Streptomyces sp. 4F.
authors: Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 20032, China.
authors: Wei-Hua
authors: WH
authors: Chen
authors: Zhong-Jun
authors: ZJ
authors: Qin
authors: Jin
authors: J
authors: Wang
authors: Guo-Ping
authors: GP
authors: Zhao
mesh_terms: D019175:DNA Methylation; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013302:Streptomyces / Q000235:genetics
chemical_list: D015252:Deoxyribonucleases, Type II Site-Specific