A high-throughput and single-tube recombination of crude PCR products using a DNA polymerase inhibitor and type IIS restriction enzyme

Abstract: Type IIS restriction enzymes have been successfully used as “universal” restriction enzymes in DNA manipulations. We took a step further to develop a rapid technique for recombining DNA fragments, fully automatic single-tube recombination (FASTR), which enables multiple-fragment DNA recombination in a single step. Crude PCR products are directly mixed with both type IIS restriction endonuclease and DNA ligase to initiate a spontaneous and one-way recombination reaction. Highly efficient DNA recombination can be achieved by an inhibition of DNA polymerase with aphidicolin and a selective digestion of template DNAs by DpnI, a restriction enzyme to digest hemi-methylated DNA in the reaction solution; thereby the entire procedure takes less than 15min. Owing to its simplicity, efficiency and rapidity, one-step FASTR can be applied to a wide range of DNA manipulations including those involving high-throughput applications where significant reduction in time and cost is expected. [Copyright &y& Elsevier]