High sensitivity mapping of methylated cytosines.
An understanding of DNA methylation and its potential role in gene control during development, aging and cancer has been hampered by a lack of sensitive methods which can resolve exact methylation patterns from only small quantities of DNA. We have now developed a genomic sequencing technique which is capable of detecting every methylated cytosine on both strands of any target sequence, using DNA isolated from fewer than 100 cells. In this method, sodium bisulphite is used to convert cytosine residues to uracil residues in single-stranded DNA, under conditions whereby 5-methylcytosine remains non-reactive. The converted DNA is amplified with specific primers and sequenced. All the cytosine residues remaining in the sequence represent previously methylated cytosines in the genome. The work described has defined procedures that maximise the efficiency of denaturation, bisulphite conversion and amplification, to permit methylation mapping of single genes from small amounts of genomic DNA, readily available from germ cells and early developmental stages.
Clark
S J
SJ
Kanematsu Laboratories, Royal Prince Alfred Hospital, Camperdown, NSW, Australia.
Harrison
J
J
Paul
C L
CL
Frommer
M
M
D044503:5-Methylcytosine; D001483:Base Sequence; D003001:Cloning, Molecular; D003596:Cytosine / Q000031:analogs & derivatives* / Q000032:analysis / Q000378:metabolism*; D017931:DNA Primers; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008745:Methylation; D008969:Molecular Sequence Data; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013447:Sulfites / Q000494:pharmacology; D014498:Uracil / Q000378:metabolism
D017931:DNA Primers; D004277:DNA, Single-Stranded; D013447:Sulfites; D014498:Uracil; D044503:5-Methylcytosine; C030981:metabisulfite; D003596:Cytosine; C009279:sodium bisulfite