Nucleic Acids Research Advance Access published online on March 17, 2009
Nucleic Acids Research, doi:10.1093/nar/gkp150
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Methods Online |
Restriction enzyme-free mutagenesis via the light regulation of DNA polymerization
Department of Chemistry, North Carolina State University, Raleigh, NC 27607-8204, USA
*To whom correspondence should be addressed. Tel: +1 919 513 2958; Fax: +1 919 515 5079; Email: alex_deiters{at}ncsu.edu
Received November 24, 2008. Revised February 17, 2009. Accepted February 20, 2009.
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.