title: In vitro assembly of multiple DNA fragments using successive hybridization.
abstract: Construction of recombinant DNA from multiple fragments is widely required in molecular biology, especially for synthetic biology purposes. Here we describe a new method, successive hybridization assembling (SHA) which can rapidly do this in a single reaction in vitro. In SHA, DNA fragments are prepared to overlap one after another, so after simple denaturation-renaturation treatment they hybridize in a successive manner and thereby assemble into a recombinant molecule. In contrast to traditional methods, SHA eliminates the need for restriction enzymes, DNA ligases and recombinases, and is sequence-independent. We first demonstrated its feasibility by constructing plasmids from 4, 6 and 8 fragments with high efficiencies, and then applied it to constructing a customized vector and two artificial pathways. As SHA is robust, easy to use and can tolerate repeat sequences, we expect it to be a powerful tool in synthetic biology.
authors.affiliation: Biomaterials Center, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, China.
authors.forename: Xinglin
authors.initials: X
authors.lastname: Jiang
authors.forename: Jianming
authors.initials: J
authors.lastname: Yang
authors.forename: Haibo
authors.initials: H
authors.lastname: Zhang
authors.forename: Huibin
authors.initials: H
authors.lastname: Zou
authors.forename: Cong
authors.initials: C
authors.lastname: Wang
authors.forename: Mo
authors.initials: M
authors.lastname: Xian
mesh_terms: D000096:Acetone / Q000378:metabolism; D019883:Alkyl and Aryl Transferases / Q000235:genetics / Q000378:metabolism; D002070:Butadienes; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004274:DNA, Recombinant / Q000235:genetics / Q000378:metabolism*; D005240:Feasibility Studies; D005822:Genetic Vectors / Q000096:biosynthesis / Q000235:genetics; D045782:Hemiterpenes / Q000096:biosynthesis; D053858:Metabolic Networks and Pathways / Q000235:genetics; D008954:Models, Biological; D009693:Nucleic Acid Hybridization / Q000379:methods*; D010420:Pentanes; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism; D017856:Phosphotransferases (Phosphate Group Acceptor) / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D002070:Butadienes; D004274:DNA, Recombinant; D045782:Hemiterpenes; D010420:Pentanes; D029701:Saccharomyces cerevisiae Proteins; C005059:isoprene; D000096:Acetone; D004247:DNA; D019883:Alkyl and Aryl Transferases; C093854:isoprene synthase; D017853:Phosphotransferases (Alcohol Group Acceptor); C021796:mevalonate kinase; D017856:Phosphotransferases (Phosphate Group Acceptor); C022371:phosphomevalonate kinase