pSpark®

From the middle of 90s to date the most popular DNA cloning system has been T/A based cloning that uses T-vectors to clone single 3´-adenine overhanging PCR products amplified with low fidelity DNA polymerases such as Taq (or Tth) DNA polymerases. T-vectors exploit the property of Taq (or Tth) DNA polymerases of non template addition of a single overhanging nucleotide, normally adenine, at the 3´-end of the polymerized DNA chain. Certain sequences called PIG sequences promote adenine addition over other nucleotides but, for unknown reasons, PIG sequences are very important only for DNA amplification of sequences longer than about 2kb for T/A based cloning. T-vectors and 3´-overhanging inserts can be joined by either T4 DNA ligase or by topoisomerase based kits available today on the market. The main benefits of T-vectors are: no extra steps after PCR, low background, flexible protocol for primers, strains and media and a trouble-free and user friendly technology.

The major drawback of T-vectors is the low fidelity of Taq polymerase that produces a high proportion of molecules with mutations. On the market there are now available second generation proofreading polymerases with fidelity up to 50 times higher than that of Taq, but cloning of blunt PCR products into T-vectors uses a very low efficiency A-tailing extra step that lowers both efficiency and throughput.