title: Realizing directional cloning using sticky ends produced by 3'-5' exonuclease of Klenow fragment.
abstract: The Klenow fragment (KF) has been used to make the blunt end as a tool enzyme. Its 5'-3' polymerase activity can extend the 5' overhanging sticky end to the blunt end, and 3'-5' exonuclease activity can cleave the 3' overhanging sticky end to the blunt end. The blunt end is useful for cloning. Here, we for the first time determined that a sticky end can be made by using the 3'-5' exonuclease activity of KF. We found that KF can cleave the blunt end into certain sticky ends under controlled conditions. We optimized enzyme cleavage conditions, and characterized the cleaved sticky ends to be mainly 2 nt 5' overhang. By using these sticky ends, we realized ligation reaction in vitro, and accomplished cloning short oligonucleotides directionally with high cloning efficiency. In some cases, this method can provide sticky end fragments in large scale for subsequent convenient cloning at low cost.
authors.affiliation: Department of Biochemistry and Molecular Biology, Preclinical Medicine College, China Medical University, Shenyang, Liaoning Province, P.R.China.
authors.forename: Guojie
authors.initials: G
authors.lastname: Zhao
authors.forename: Jun
authors.initials: J
authors.lastname: Li
authors.forename: Tianyu
authors.initials: T
authors.lastname: Hu
authors.forename: Hua
authors.initials: H
authors.lastname: Wei
authors.forename: Yifu
authors.initials: Y
authors.lastname: Guan
mesh_terms: D001483:Base Sequence; D003001:Cloning, Molecular; D004256:DNA Polymerase I / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D004277:DNA, Single-Stranded / Q000737:chemistry*; D004926:Escherichia coli; D005092:Exonucleases / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D009841:Oligonucleotides / Q000737:chemistry
chemical_list: D004277:DNA, Single-Stranded; D009841:Oligonucleotides; D004256:DNA Polymerase I; D005092:Exonucleases