title: A simple and convenient sticky/blunt-end ligation method for fusion gene construction.
abstract: Here we present a simple and convenient sticky/blunt-end ligation method for fusion gene construction. The fusion gene is constructed by seamless ligation of 5'-end phosphorylated blunt ends instead of by overlap extension PCR (OE-PCR). Therefore, the challenge of amplifying large DNA fragments (e.g., the large bifunctional enzyme gene constructed by fusion of two monofunctional enzyme genes) by PCR can be avoided. In addition, synthesis of the inner primers for OE-PCR is not necessary, indicating that this method should be especially convenient for construction of fusion genes with various combinations of multiple fragments (e.g., chimeric gene libraries, fusion gene libraries). As a modification of the commonly used fusion gene construction technique, this method may find a wide range of applications in bioscience and biotechnology.
authors.affiliation: College of Biosciences and Biotechnology, Shenyang Agricultural University, No. 120 Dongling Road, Shenyang, 110161, People's Republic of China.
authors.forename: Song
authors.initials: S
authors.lastname: Gao
authors.forename: Jiannan
authors.initials: J
authors.lastname: Zhang
authors.forename: Tianjin
authors.initials: T
authors.lastname: Miao
authors.forename: Di
authors.initials: D
authors.lastname: Ma
authors.forename: Ying
authors.initials: Y
authors.lastname: Su
authors.forename: Yingfeng
authors.initials: Y
authors.lastname: An
authors.forename: Qingrui
authors.initials: Q
authors.lastname: Zhang
mesh_terms: D017122:Bacteriophage T4 / Q000737:chemistry / Q000201:enzymology; D001483:Base Sequence; D003001:Cloning, Molecular; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000235:genetics* / Q000378:metabolism; D017931:DNA Primers / Q000138:chemical synthesis / Q000235:genetics*; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D015723:Gene Library; D008969:Molecular Sequence Data; D010767:Phosphorylcholine; D016133:Polymerase Chain Reaction / Q000379:methods*; D011116:Polynucleotide 5'-Hydroxyl-Kinase / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics*; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D010767:Phosphorylcholine; D011116:Polynucleotide 5'-Hydroxyl-Kinase; D011088:DNA Ligases; D000072481:DNA Ligase ATP