Fluorometric identification of 5-methylcytosine modification in DNA: combination of photosensitized oxidation and invasive cleavage.
An efficient fluorometric detection system of DNA methylation has been developed by a combination of a photooxidative DNA cleavage reaction with 2-methyl-1,4-naphthoquinone (NQ) chromophore and an invasive cleavage reaction with human Flap endonuclease-1. Enzymatic treatment of a mixture of photochemically fragmented target oligodeoxynucleotides (ODNs) at 5-methylcytosine mC) and hairpin-like probe oligomer possessing a fluorophore (F) and a quencher (D) resulted in a dramatic enhancement of fluorescence. In contrast, fluorescence emission for the ODN containing cytosine but not mC at the target sequence was extremely weak. In addition, by monitoring the fluorescence change, this system allows for the detection of mC in DNA at subfemtomole amounts. This system would provide a highly sensitive protocol for determining the methylation status in DNA by fluorescence emission.
Department of Energy and Hydrocarbon Chemistry, Graduate School of Engineering, Kyoto University, Katsura Campus, Kyoto 615-8510, Japan.
Hisatsugu
H
Yamada
Kazuhito
K
Tanabe
Sei-ichi
S
Nishimoto
D044503:5-Methylcytosine / Q000737:chemistry*; D001483:Base Sequence; D053837:DNA Cleavage; D019175:DNA Methylation; D045585:Flap Endonucleases / Q000378:metabolism; D005470:Fluorometry / Q000379:methods*; D006801:Humans; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism; D010084:Oxidation-Reduction; D010777:Photochemistry; D013696:Temperature; D024483:Vitamin K 3 / Q000737:chemistry
D009841:Oligonucleotides; D044503:5-Methylcytosine; D024483:Vitamin K 3; D045585:Flap Endonucleases