Restriction enzyme-free mutagenesis via the light regulation of DNA polymerization.
The effects of photocaged nucleosides on the DNA polymerization reaction was investigated, finding that most polymerases are unable to recognize and read through the presence of a single caging group on the DNA template. Based on this discovery, a new method of introducing mutations into plasmid DNA via a light-mediated mutagenesis protocol was developed. This methodology is advantageous over several common approaches in that it requires the use of only two polymerase chain reaction primers, and does not require any restriction sites or use of restriction enzymes. Additionally, this approach enables not only site-directed mutations, but also the insertion of DNA strands of any length into plasmids and the deletion of entire genes from plasmids.
Young
Douglas D
DD
Department of Chemistry, North Carolina State University, Raleigh, NC 27607-8204, USA.
Lusic
Hrvoje
H
Lively
Mark O
MO
Deiters
Alexander
A
D052117:Benzodioxoles / Q000737:chemistry; D003001:Cloning, Molecular; D004247:DNA / Q000096:biosynthesis / Q000737:chemistry / Q000528:radiation effects*; D017931:DNA Primers; D004262:DNA Restriction Enzymes; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D009705:Nucleosides / Q000737:chemistry; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D017384:Sequence Deletion; D013698:Templates, Genetic; D014466:Ultraviolet Rays
D052117:Benzodioxoles; D017931:DNA Primers; D009705:Nucleosides; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D004262:DNA Restriction Enzymes