Regular Article
A Sensitive New Method for Rapid Detection of Abnormal Methylation Patterns in Global DNA and within CpG Islands

https://doi.org/10.1006/bbrc.1999.1187Get rights and content

Abstract

To assess alterations in DNA methylation density in both global DNA and within CpG islands, we have developed a simple method based on the use of methylation-sensitive restriction endonucleases that leave a 5′ guanine overhang after DNA cleavage, with subsequent single nucleotide extension with radiolabeled [3H]dCTP. The methylation-sensitive restriction enzymes HpaII and AciI have relatively frequent recognition sequences at CpG sites that occur randomly throughout the genome. BssHII is a methylation sensitive enzyme that similarly leaves a guanine overhang, but the recognition sequence is nonrandom and occurs predominantly at unmethylated CpG sites within CpG islands. The selective use of these enzymes can be used to screen for alterations in genome-wide methylation and CpG island methylation status, respectively. The extent of [3H]dCTP incorporation opposite the exposed guanine after restriction enzyme treatment is directly proportional to the number of unmethylated (cleaved) CpG sites. The “cytosine-extension assay” has several advantages over existing methods because (a) radiolabel incorporation is independent of the integrity of the DNA, (b) methylation detection does not require PCR amplification or DNA methylase reactions, and (c) it is applicable to ng quantities of DNA. Using DNA extracted from normal human liver and from human hepatocellular carcinoma, the applicability of the assay is demonstrated by the detection of an increase in genome-wide hypomethylation and CpG island hypermethylation in the tumor DNA.

References (22)

  • M. Balaghi et al.

    Biochem. Biophys. Res. Commun.

    (1993)
  • I.P. Pogribny et al.

    Cancer Lett.

    (1997)
  • J. Bender

    TIBS

    (1998)
  • E. Wainfan et al.

    Cancer Res.

    (1992)
  • J. Soares et al.

    Cancer

    (1999)
  • K.L. Steinmetz et al.

    Carcinogenesis

    (1998)
  • I.P. Pogribny et al.

    Cancer Res.

    (1995)
  • S.A. Belinsky et al.

    Proc. Natl. Acad. Sci. USA

    (1996)
  • J.P.J. Issa et al.

    Cancer Res.

    (1996)
  • L. Sun et al.

    Jap. J. Cancer Res.

    (1997)
There are more references available in the full text version of this article.

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To whom correspondence should be addressed at Division of Biochemical Toxicology, NCTR, 3900 NCTR Road, Jefferson, AR 72079. Fax: (870) 543-6620. E-mail: [email protected].

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