METHODS, COMPOSITIONS, AND KITS FOR ONE-STEP DNA CLONING USING DNA TOPOISOMERASE

摘要

Provided herein are methods, compositions, kits, and vectors useful for molecular cloning of, for example, blunt-ended DNA molec using DNA topoisomerase. In certain embodiments, the methods comprise combining into a mixture a first polynucleotide, wherein first polynucleotide comprises an origin of replication, a selectable marker, two topoisomerase recognition sequences, and two nick agent recognition sequences, wherein each of said two topoisomerase recognition sequences is within 0-50 nucleotides of at least of the nicking agent recognition sequences and wherein each of said two nicking agent recognition sequences is nicked, with a sequence-specific topoisomerase and a second polynucleotide, wherein said second polynucleotide comprises a 5' hydroxyl on ea end of the polynucleotide; and transforming the mixture into a host organism, thereby cloning the second polynucleotide.