Identification and characterization of differentially methylated regions of genomic DNA by methylation sensitive arbitrarily primed PCR

M. L. Gonzalyo, C. H. Spruck, J. M. Zingg, A. S. Yang, P. A. Jones

Research output: Contribution to journalArticlepeer-review

Abstract

The methylation of cytosine at CpG dinucleotides in vertebrates is an epigenetic modification which may contribute to tumorigenesis via generation of point mutations or alteration of gene expression. Clusters of CpG dinucleotides form what are known as CpG islands in the 5′ region of many genes and may serve to regulate the transcriptional activity of these elements. Consequently, changes in the methylation status of CpG islands may alter the expression of any number of genes that are associated with cellular transformation. Hypomethylation is usually associated with gene activity, while hypermethylation of CpG islands may cause gene inactivation. We have developed a methylation sensitive arbitrarily primed PCR (AP-PCR) technique that can be used, in a reproducible manner, for the rapid identification and characterization of specific regions of genomic DNA that have undergone methylation changes associated with processes such as transformation. An AP-PCR assay based on the insensitivity of some restriction enzymes to cut methylated sequences was utilized to determine changes in DNA methylation at various sites in human genomic DNA in normal and tumor colon tissues from ten patients and six bladder cancer cell lines. After amplification by AP-PCR, differentially methylated fragments were identified, cloned, and sequenced. Both hyper- and hypomethylation of specific regions of tumor DNA compared to normal tissues was observed. Southern analysis of restriction-digested genomic DNA was performed to confirm differentially methylated events identified by this technique. Methylation sensitive AP-PCR can be used as a powerful assay for evaluating methylation differences at several unique sites in a rapid and efficient manner. Early detection of methylation changes associated with tumongenesis may be potentially useful for the diagnosis and treatment of specific cancer types.

Original languageEnglish (US)
Pages (from-to)105A
JournalJournal of Investigative Medicine
Volume44
Issue number1
StatePublished - 1996
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

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