Directional TOPO® Cloning
|Directional TOPO® cloning enables cloning of blunt-ended PCR products in a 5´→3´ orientation directly into a expression vector using a 5-minute ligation reaction, thereby eliminating subcloning steps and saving you time. Directional TOPO® cloning vectors contain a single-strand GTGG overhang on the 5´ end and a blunt end on the 3´ end. The four-nucleotide overhang invades the double-strand DNA of the PCR product and anneals to the |
CACC sequence that you place in your 5´ primer. Topoisomerase I then ligates the PCR product in the correct orientation.
With Directional TOPO® Cloning Expression Kits, you will:
Figure 1: Directional cloning of human open reading frames into pcDNA® 3.1 D/VS- His- TOPO® vector.