Urea improves efficiency of bisulphite-mediated sequencing of 5'-methylcytosine in genomic DNA.
The detection of 5'-methylcytosine by the bisulphite-mediated genomic sequencing method has considerably aided study of the role of methylation in areas such as X chromosome inactivation, genomic imprinting and cancer research. However on occasion difficulty has been experienced in obtaining complete conversion of cytosine to uracil in regions of the target DNA. We report here a simple improvement to the method involving addition of urea to the bisulphite reaction, a step which greatly improves the reaction efficiency, presumably by maintaining the target DNA in single stranded form, thereby allowing complete and reliable conversion.
Department of Cell and Molecular Biology, University of Technology, Sydney, Westbourne Street, Gore Hill, NSW 2065, Australia.
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Paulin
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Grigg
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Davey
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Piper
D044503:5-Methylcytosine; D001483:Base Sequence; D003596:Cytosine / Q000031:analogs & derivatives* / Q000737:chemistry; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000235:genetics*; D019450:Genes, MDR; D015894:Genome, Human; D006801:Humans; D007202:Indicators and Reagents; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000379:methods*; D013447:Sulfites; D014508:Urea
D004277:DNA, Single-Stranded; D007202:Indicators and Reagents; D013447:Sulfites; D044503:5-Methylcytosine; D003596:Cytosine; D014508:Urea