Chemical restriction: strand cleavage by ammonia treatment at 8-oxoguanine yields biologically active DNA.
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 degrees 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.
Meyer
Christoph
C
Department of Chemistry, University of Basel, St. Johanns-Ring 19, 4056 Basel, Switzerland.
Meyer
Dominik
D
Bickle
Thomas A
TA
Giese
Bernd
B
D000641:Ammonia / Q000737:chemistry*; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D017931:DNA Primers / Q000737:chemistry / Q000235:genetics; D004274:DNA, Recombinant / Q000138:chemical synthesis*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D006147:Guanine / Q000031:analogs & derivatives* / Q000737:chemistry*; D009841:Oligonucleotides / Q000737:chemistry; D016133:Polymerase Chain Reaction
D017931:DNA Primers; D004274:DNA, Recombinant; D009841:Oligonucleotides; C024829:8-hydroxyguanine; D006147:Guanine; D000641:Ammonia; D004259:DNA-Directed DNA Polymerase