Construction of T-vector derived from pBluescript ΙΙ SK with a positive selection marker, a rapid system for cloning

Document Type: Original Research Papers

Authors

1 Sari Agricultural Sciences and Natural Resources University

2 Genetics and Agricultural Biotechnology Institute of Tabarestan, Sari Agricultural Sciences and Natural Resources University, Sari, Mazandaran

Abstract

A rapid DNA cloning system is a research interest of many scientists. TA cloning is one of the methods used for the cloning of PCR-amplified DNA molecules. The TA cloning method is a convenient and labor-saving replacement to traditional, restriction enzyme-mediated cloning strategies. A T-vector called pBlueskript ΙΙ SK-1 with the lethal gene ccdB was designed to construct a positive selection vector. This lethal gene was inserted in multiple cloning sites of pBlueskript ΙΙ SK. Then the vector digested with the endonuclease SmaΙ producing the blunt end. To directly clone the PCR product, a single 3'-A was added to a double-stranded DNA fragment by Taq polymerase and a T-vector with 3'-T overhang at each end using ddTTP and terminal transferase enzyme. The recombinant vector was transferred to the competent cells of host Escherichia coli. After DNA fragment entry, the activity of the ccdB gene eliminated, and the survival probability and host colony formation increased after transformation with the recombinant vector. The proliferation of the host of the T-vector was highly specific, and only hosts with the ccdA gene were able to receive this vector, to replicate the vector and survive. Therefore, after the insertion of the target gene, the lethal gene becomes inactivated, so there was no need to use a specific host and other selective markers, such as antibiotics. The TA cloning with a positive selection marker strategy is both simple and much more efficient than blunt-ended ligation and cohesive-end cloning.

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