High-speed conversion of cytosine to uracil in bisulfite genomic sequencing analysis of DNA methylation.
Bisulfite genomic sequencing is a widely used technique for analyzing cytosine-methylation of DNA. By treating DNA with bisulfite, cytosine residues are deaminated to uracil, while leaving 5-methylcytosine largely intact. Subsequent PCR and nucleotide sequence analysis permit unequivocal determination of the methylation status at cytosine residues. A major caveat associated with the currently practiced procedure is that it takes 16-20 hr for completion of the conversion of cytosine to uracil. Here we report that a complete deamination of cytosine to uracil can be achieved in shorter periods by using a highly concentrated bisulfite solution at an elevated temperature. Time course experiments demonstrated that treating DNA with 9 M bisulfite for 20 min at 90 degrees C or 40 min at 70 degrees C all cytosine residues in the DNA were converted to uracil. Under these conditions, the majority of 5-methylcytosines remained intact. When a high molecular weight DNA derived from a cell line (containing a number of genes whose methylation status was known) was treated with bisulfite under the above conditions and amplified and sequenced, the results obtained were consistent with those reported in the literature. Although some degradation of DNA occurred during this process, the amount of treated DNA required for the amplification was nearly equal to that required for the conventional bisulfite genomic sequencing procedure. The increased speed of DNA methylation analysis with this novel procedure is expected to advance various aspects of DNA sciences.
Shiraishi
Masahiko
M
DNA Methylation and Genome Function Project, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan. mshirais@gan2.res.ncc.go.jp
Hayatsu
Hikoya
H
D001483:Base Sequence; D045744:Cell Line, Tumor; D018899:CpG Islands; D003596:Cytosine / Q000737:chemistry*; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D019175:DNA Methylation; D005821:Genetic Techniques; D006801:Humans; D008957:Models, Genetic; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000379:methods*; D013447:Sulfites / Q000494:pharmacology*; D013696:Temperature; D013997:Time Factors; D014498:Uracil / Q000737:chemistry*
D013447:Sulfites; D014498:Uracil; D003596:Cytosine; D004247:DNA