Random DNA fragmentation with endonuclease V: application to DNA shuffling.
The enzyme endonuclease V nicks uracil-containing DNA at the second or third phosphodiester bond 3' to uracil sites. I applied the enzyme to random fragmentation of DNA to revise the complex DNA shuffling protocol. The merit of using endonuclease V is that cleavage occurs at random sites and the length of the fragments can easily be adjusted by varying the concentration of dUTP in the polymerase chain reaction. Unlike the conventional method using DNase I, no partial digestion or gel separation of fragments is required. Therefore, labor is dramatically reduced and reproducibility ensured. I applied this method to recombine two truncated green fluorescent protein (GFP) genes and demonstrated successful DNA shuffling by the appearance of the fluorescent full-length GFP genes.
Miyazaki
Kentaro
K
Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan. miyazaki-kentaro@aist.go.jp
D004247:DNA / Q000235:genetics / Q000378:metabolism*; D043224:Deoxyribonuclease (Pyrimidine Dimer); D003854:Deoxyribonucleotides / Q000378:metabolism; D003856:Deoxyuracil Nucleotides / Q000378:metabolism; D019020:Directed Molecular Evolution / Q000379:methods; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015723:Gene Library; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results
D003854:Deoxyribonucleotides; D003856:Deoxyuracil Nucleotides; D011993:Recombinant Fusion Proteins; D004247:DNA; D004706:Endodeoxyribonucleases; D043224:Deoxyribonuclease (Pyrimidine Dimer)