Young Douglas D, Lusic Hrvoje, Lively Mark O, Deiters Alexander
Department of Chemistry, North Carolina State University, Raleigh, NC 27607-8204, USA.
Nucleic Acids Res. 2009 May;37(8):e58. doi: 10.1093/nar/gkp150. Epub 2009 Mar 17.
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.
研究了光笼化核苷对DNA聚合反应的影响，发现大多数聚合酶无法识别并通读DNA模板上单个笼化基团的存在。基于这一发现，开发了一种通过光介导诱变方案在质粒DNA中引入突变的新方法。该方法优于几种常见方法，因为它只需要使用两个聚合酶链反应引物，不需要任何限制性酶切位点或使用限制性酶。此外，这种方法不仅能够实现定点突变，还能将任何长度的DNA链插入质粒，并从质粒中删除整个基因。
