Search tips
Search criteria 


Logo of narLink to Publisher's site
Nucleic Acids Res. 1994 August 11; 22(15): 2990–2997.
PMCID: PMC310266

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.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (1.9M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Images in this article

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Singer J, Roberts-Ems J, Riggs AD. Methylation of mouse liver DNA studied by means of the restriction enzymes msp I and hpa II. Science. 1979 Mar 9;203(4384):1019–1021. [PubMed]
  • Singer-Sam J, Grant M, LeBon JM, Okuyama K, Chapman V, Monk M, Riggs AD. Use of a HpaII-polymerase chain reaction assay to study DNA methylation in the Pgk-1 CpG island of mouse embryos at the time of X-chromosome inactivation. Mol Cell Biol. 1990 Sep;10(9):4987–4989. [PMC free article] [PubMed]
  • Saluz H, Jost JP. A simple high-resolution procedure to study DNA methylation and in vivo DNA-protein interactions on a single-copy gene level in higher eukaryotes. Proc Natl Acad Sci U S A. 1989 Apr;86(8):2602–2606. [PubMed]
  • Pfeifer GP, Steigerwald SD, Mueller PR, Wold B, Riggs AD. Genomic sequencing and methylation analysis by ligation mediated PCR. Science. 1989 Nov 10;246(4931):810–813. [PubMed]
  • Maxam AM, Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. [PubMed]
  • Church GM, Gilbert W. Genomic sequencing. Proc Natl Acad Sci U S A. 1984 Apr;81(7):1991–1995. [PubMed]
  • Frommer M, McDonald LE, Millar DS, Collis CM, Watt F, Grigg GW, Molloy PL, Paul CL. A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc Natl Acad Sci U S A. 1992 Mar 1;89(5):1827–1831. [PubMed]
  • Wang RY, Gehrke CW, Ehrlich M. Comparison of bisulfite modification of 5-methyldeoxycytidine and deoxycytidine residues. Nucleic Acids Res. 1980 Oct 24;8(20):4777–4790. [PMC free article] [PubMed]
  • Shapiro R, Braverman B, Louis JB, Servis RE. Nucleic acid reactivity and conformation. II. Reaction of cytosine and uracil with sodium bisulfite. J Biol Chem. 1973 Jun 10;248(11):4060–4064. [PubMed]
  • Shapiro R, DiFate V, Welcher M. Deamination of cytosine derivatives by bisulfite. Mechanism of the reaction. J Am Chem Soc. 1974 Feb 6;96(3):906–912. [PubMed]
  • Hayatsu H. Bisulfite modification of nucleic acids and their constituents. Prog Nucleic Acid Res Mol Biol. 1976;16:75–124. [PubMed]
  • Harrison J, Molloy PL, Clark SJ. Direct cloning of polymerase chain reaction products in an XcmI T-vector. Anal Biochem. 1994 Jan;216(1):235–236. [PubMed]
  • Tasheva ES, Roufa DJ. Deoxycytidine methylation and the origin of spontaneous transition mutations in mammalian cells. Somat Cell Mol Genet. 1993 May;19(3):275–283. [PubMed]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press