Random DNA fragmentation with endonuclease V: application to DNA shuffling | Zendy

Kentaro Miyazaki | Zendy

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Random DNA fragmentation with endonuclease V: application to DNA shuffling

Author(s) -

Kentaro Miyazaki

Publication year - 2002

Publication title -

nucleic acids research

Language(s) - English

Resource type - Journals

SCImago Journal Rank - 9.008

H-Index - 537

eISSN - 1362-4954

pISSN - 0305-1048

DOI - 10.1093/nar/gnf139

Subject(s) - biology , endonuclease , microbiology and biotechnology , dna , restriction enzyme , dna polymerase , dna clamp , dna polymerase ii , fragmentation (computing) , in vitro recombination , uracil , phosphodiester bond , dna shuffling , biochemistry , polymerase chain reaction , gene , molecular cloning , complementary dna , mutant , directed evolution , rna , reverse transcriptase , ecology

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.

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