title: Hot Fusion: an efficient method to clone multiple DNA fragments as well as inverted repeats without ligase.
abstract: Molecular cloning is utilized in nearly every facet of biological and medical research. We have developed a method, termed Hot Fusion, to efficiently clone one or multiple DNA fragments into plasmid vectors without the use of ligase. The method is directional, produces seamless junctions and is not dependent on the availability of restriction sites for inserts. Fragments are assembled based on shared homology regions of 17-30 bp at the junctions, which greatly simplifies the construct design. Hot Fusion is carried out in a one-step, single tube reaction at 50 °C for one hour followed by cooling to room temperature. In addition to its utility for multi-fragment assembly Hot Fusion provides a highly efficient method for cloning DNA fragments containing inverted repeats for applications such as RNAi. The overall cloning efficiency is in the order of 90-95%.
authors.affiliation: Biotechnology, Monsanto Company, St. Louis, Missouri, United States of America.
authors.forename: Changlin
authors.initials: C
authors.lastname: Fu
authors.affiliation: Biotechnology, Monsanto Company, St. Louis, Missouri, United States of America.
authors.forename: William P
authors.initials: WP
authors.lastname: Donovan
authors.affiliation: Biotechnology, Monsanto Company, St. Louis, Missouri, United States of America.
authors.forename: Olga
authors.initials: O
authors.lastname: Shikapwashya-Hasser
authors.affiliation: Biotechnology, Monsanto Company, St. Louis, Missouri, United States of America.
authors.forename: Xudong
authors.initials: X
authors.lastname: Ye
authors.affiliation: Biotechnology, Monsanto Company, St. Louis, Missouri, United States of America.
authors.forename: Robert H
authors.initials: RH
authors.lastname: Cole
mesh_terms: D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000737:chemistry* / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D017343:Genes, Plant / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D055029:Inverted Repeat Sequences; D008025:Ligases / Q000378:metabolism; D016296:Mutagenesis; D010957:Plasmids / Q000235:genetics; D034622:RNA Interference; D013997:Time Factors
chemical_list: D004247:DNA; D008025:Ligases