Sensitive discrimination between cytosine and 5-methylcytosine in DNA by a modified invader method.
We designed a detection system of 5-methylcytosine ((m)C) in DNA by a combination of photooxidative DNA cleavage reaction of 2-methyl-1,4-naphthoquinone chromophore with invader technology. Enzymatic treatment of a mixture of photochemically fragmented target oligodeoxynucleotides (ODNs) at (m)C and probe oligomer possessing a fluorophore and a quencher resulted in a dramatic enhancement of fluorescence. In contrast, fluorescence emission for the ODN containing C but not (m)C at the target sequence was extremely weak. We could detect (m)C in sub-femtomol DNA by monitoring the fluorescence change.
Yamada
Hisatsugu
H
Department of Energy and Hydrocarbon Chemistry, Graduate School of Engineering, Kyoto University, Katsura Campus, Kyoto 615-8510, Japan.
Tanabe
Kazuhito
K
Nishimoto
Sei-ichi
S
D044503:5-Methylcytosine / Q000032:analysis*; D003596:Cytosine / Q000032:analysis; D004247:DNA / Q000737:chemistry; D045585:Flap Endonucleases; D005456:Fluorescent Dyes / Q000737:chemistry*; D009693:Nucleic Acid Hybridization / Q000379:methods*; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000528:radiation effects; D015345:Oligonucleotide Probes / Q000737:chemistry*; D010777:Photochemistry; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes; D009838:Oligodeoxyribonucleotides; D015345:Oligonucleotide Probes; D044503:5-Methylcytosine; D003596:Cytosine; D004247:DNA; D045585:Flap Endonucleases