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Chemical Restriction: Strand Cleavage by Ammonia Treatment at 8‐Oxoguanine Yields Biologically Active DNA
Author(s) -
Meyer Christoph,
Meyer Dominik,
Bickle Thomas A.,
Giese Bernd
Publication year - 2003
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.200300587
Subject(s) - dna , restriction enzyme , cleavage (geology) , plasmid , oligonucleotide , microbiology and biotechnology , chemistry , multiple cloning site , restriction site , cloning (programming) , gene , biology , biochemistry , recombinant dna , vector (molecular biology) , paleontology , fracture (geology) , computer science , programming language
Cleavage of DNA single and double strands at an 8‐oxoguanine‐containing nucleotide occurs in 90 % yield if the modified oligonucleotide is treated with NH 3 and O 2 at 60 °C. The mechanism of this oxidative cleavage reaction was studied, and the reaction was applied to the generation of single‐stranded overhangs on PCR‐amplified DNA that can be ligated. As an example, the lac Z′ gene was amplified by PCR with 8‐oxoguanine modified primers, restricted by ammonia treatment, ligated into a plasmid vector, transformed in Escherischia coli cells, and screened for blue colonies. This method guarantees efficiencies comparable to the standard cloning procedure with restriction enzymes, and it allows the design of any 3′‐overhang independent of the sequence of the cloned DNA.