> DNA Methylation analysis PCR Primer Database


Alterations in the patterns of DNA methylation are among the earliest and most common events in tumorigenesis (1). In the mammalian genome, methylation takes place only at cytosine bases that are located 5' to a guanosine in a CpG dinucleotide. While this dinucleotide is generally underrepresented in the genome, short regions are rich in CpG content. Many gene promoters contain CpG-rich regions of DNA that are known as CpG islands (2). Both global hypomethylation and regional hypermethylation have been described in human tumor cell lines and a wide spectrum of cancers (3). Hypomethylation (or absence of methylation) of CpG islands is typically associated with gene activity, while hypermethylation of these promoter-containing CpG islands has been correlated with decreased gene activity (4). The development of efficient and accurate methods to study cytosine methylation is therefore of critical importance in understanding the role of DNA methylation in the development and progression of cancer.

 DNA methylation analysis

Several techniques have been described for evaluation of cytosine methylation including digestion of DNA with methylation-sensitive restriction enzymes followed by Southern blotting or polymerase chain reaction (PCR) (5). Southern blotting requires large amounts of high molecular weight DNA, which limits the use of this technique. The above mentioned limitations are counteracted by performing PCR, but still both methods rely on a complete enzymatic digestion of the DNA in order to prevent false-positive results.

 DNA methylation analysis methods

Instead of using methylation-sensitive restriction enzymes, other methods are based on sodium bisulfite treatment of the DNA to introduce methylation-dependent sequence differences into the genomic DNA. Sodium bisulfite converts unmethylated cytosine to uracil while leaving 5-methylcytosine unchanged. Nowadays, the most frequently used DNA methylation analysis methods employ a combination of bisulfite treatment and PCR.

Methylation-sensitive single-nucleotide primer extension

The methylation-sensitive single-nucleotide primer extension (Ms-SNuPE) method incorporates amplification of bisulfite-treated DNA, followed by a quantification of the ratio of methylated versus unmethylated cytosines at CpG sites (6). This is accomplished by incubating the PCR product with internal primers that anneal to the PCR template and terminate immediately 5' of the cytosine to be assayed. The relative amount of methylation is quantitated by measuring the incorporation of labelled dCTP or dTTP. Several CpG sites can be analysed in a single reaction by using different-length primers for each site. Ms-SNuPE however is considered to be relatively labor intensive for screening applications.

Combined bisulfite restriction analysis

An alternative method, called combined bisulfite restriction analysis (COBRA), uses standard sodium bisulfite PCR treatment followed by restriction digestion and band quantification (7). The primers used in the PCR do not span CpG dinucleotides so that the amplification step does not discriminate between templates according to their original methylation status. Instead, restriction enzymes are used to indicate methylation, based on the creation of new or retention of pre-existing restriction sites after bisulfite modification. The fraction digested/uncut PCR product is a direct reflection of the percentage DNA methylation at that site in the original genomic DNA.


A more widespread procedure combines a bisulfite treatment and PCR-single-strand conformation polymorphism analysis (Bisulfite-PCR-SSCP or BiPS) (8). In a first step, the converted DNA is amplified with primers that have no CpG sites in the corresponding region of the original DNA, and as such amplify both unmethylated and methylated DNA. Sequence differences between amplified products from unmethylated and methylated DNA are visualised on a SSCP gel. BiPS analysis is not always straightforward because more than two bands or a smear can be observed. These unfavourable patterns hinder accurate quantification, and therefore PCR primer design and the electrophoretic conditions should be thoroughly optimised.

Methylation-specific PCR

The fourth and most popular method is methylation-specific PCR (9). It is a breakthrough in speed and sensitivity for gene methylation analysis. After bisulfite conversion, PCR is performed using primers that distinguish methylated from unmethylated DNA. Unlike the procedures using restriction enzymes, MSP can be used to analyse any specific CpG site by appropriate primer design and it is not prone to false-positive results. MSP is very sensitive, permitting the analysis of small and heterogeneous samples, including paraffin-embedded material. In addition to the primer sets for unmethylated and methylated DNA which has undergone a chemical modification, a third primer set can be used that anneals to any DNA sequence (unmethylated or methylated) that has not undergone chemical modification. This reaction is optional but serves as a control for the success of the bisulfite treatment.

Bisulfite sequencing

Sequencing bisulfite-altered DNA is the most straightforward way to detect and locate cytosine methylation (10). After denaturation and bisulfite modification, the fragment of interest is amplified by PCR, generally by primers that make no distinction between methylated and unmethylated alleles. For the sequencing of the PCR product, two strategies are available. A first one involves cloning of the PCR product into plasmid vectors, followed by sequencing individual clones, which provides methylation maps of single DNA molecules. A second strategy employs direct sequencing, which results in assessment of the average methylation status in the cells under investigation. Quantification of the cytosine methylation by direct sequencing is possible by performing peak height analysis (11).


  1. Bird, A. (2002) DNA methylation patterns and epigenetic memory. Genes Dev, 16, 6-21. (abstract)
  2. Liu, Z.J. and Maekawa, M. (2003) Polymerase chain reaction-based methods of DNA methylation analysis. Anal Biochem, 317, 259-265. (abstract)
  3. Esteller, M., Sanchez-Cespedes, M., Rosell, R., Sidransky, D., Baylin, S.B. and Herman, J.G. (1999) Detection of aberrant promoter hypermethylation of tumor suppressor genes in serum DNA from non-small cell lung cancer patients. Cancer Res, 59, 67-70. (abstract)
  4. Jones, P.A. and Baylin, S.B. (2002) The fundamental role of epigenetic events in cancer. Nat Rev Genet, 3, 415-428. (abstract)
  5. Fraga, M.F. and Esteller, M. (2002) DNA methylation: a profile of methods and applications. Biotechniques, 33, 632, 634, 636-649. (abstract)
  6. Gonzalgo, M.L. and Jones, P.A. (1997) Rapid quantitation of methylation differences at specific sites using methylation-sensitive single nucleotide primer extension (Ms-SNuPE). Nucleic Acids Res, 25, 2529-2531. (abstract)
  7. Xiong, Z. and Laird, P.W. (1997) COBRA: a sensitive and quantitative DNA methylation assay. Nucleic Acids Res, 25, 2532-2534. (abstract)
  8. Maekawa, M., Sugano, K., Kashiwabara, H., Ushiama, M., Fujita, S., Yoshimori, M. and Kakizoe, T. (1999) DNA methylation analysis using bisulfite treatment and PCR-single-strand conformation polymorphism in colorectal cancer showing microsatellite instability. Biochem Biophys Res Commun, 262, 671-676. (abstract)
  9. Herman, J.G., Graff, J.R., Myohanen, S., Nelkin, B.D. and Baylin, S.B. (1996) Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci U S A, 93, 9821-9826. (abstract)
  10. Frommer, M., McDonald, L.E., Millar, D.S., Collis, C.M., Watt, F., Grigg, G.W., Molloy, P.L., Paul, C.L. (1992) A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc Natl Acad Sci U S A, 89, 1827-1831. (abstract)
  11. Paul, C.L., Clark, S.J. (1996) Cytosine methylation: quantitation by automated genomic sequencing and GENESCAN analysis. Biotechniques, 21, 126-133. (abstract)

Ghent University, Faculty of Medicine and Health Sciences, Centre for Medical Genetics
1K5, De Pintelaan 185, B-9000 Gent, Belgium
Tel.: +32 9 240 55 18 - fax: +32 9 240 49 70 - Webmaster - Disclaimer & copyright
Valid HTML 4.01! Valid CSS!