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

J Biotechnol. 2008 Oct 10;137(1-4):1-7. doi: 10.1016/j.jbiotec.2008.07.1816. Epub 2008 Jul 23.


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 15 min. 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aphidicolin / pharmacology*
  • Cloning, Molecular / methods*
  • DNA / metabolism
  • DNA Ligases / metabolism
  • Deoxyribonucleases, Type II Site-Specific / metabolism*
  • Enzyme Inhibitors / pharmacology*
  • Nucleic Acid Synthesis Inhibitors
  • Polymerase Chain Reaction / methods*
  • Spectrometry, Fluorescence / methods


  • Enzyme Inhibitors
  • Nucleic Acid Synthesis Inhibitors
  • Aphidicolin
  • DNA
  • endodeoxyribonuclease DpnI
  • Deoxyribonucleases, Type II Site-Specific
  • DNA Ligases