| 摘要 |
Provided herein are methods, compositions, and kits useful for molecular cloning of, for example, blunt-ended DNA molecules using DNA topoisomerase. In certain embodiments, the methods comprise combining into a mixture a first polynucleotide, wherein said first polynucleotide comprises an origin of replication, a selectable marker, two topoisomerase recognition sequences, and two nicking agent recognition sequences, wherein each of said two topoisomerase recognition sequences is within 0-50 nucleotides of at least one 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 each end of the polynucleotide; and transforming the mixture into a host organism, thereby cloning the second polynucleotide. Advantageously, in some embodiments, the methods, compositions, and kits does not require the formation or purification of a DNA-protein adduct prior to the addition of the polynucleotide to be cloned. Also provided are vector sequences to facilitate performance of the methods and methods for modifying a vector of interest to render it useful in the methods described herein.
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