A high-throughput and single-tube recombination of crude PCR products using a DNA polymerase inhibitor and type IIS restriction enzyme.
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.
Kotera
Ippei
I
PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho Kawaguchi, Saitama 332-0012, Japan.
Nagai
Takeharu
T
D016590:Aphidicolin / Q000494:pharmacology*; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000378:metabolism; D011088:DNA Ligases / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D019384:Nucleic Acid Synthesis Inhibitors; D016133:Polymerase Chain Reaction / Q000379:methods*; D013050:Spectrometry, Fluorescence / Q000379:methods
D004791:Enzyme Inhibitors; D019384:Nucleic Acid Synthesis Inhibitors; D016590:Aphidicolin; D004247:DNA; C020102:endodeoxyribonuclease DpnI; D015252:Deoxyribonucleases, Type II Site-Specific; D011088:DNA Ligases