MethylScreen: DNA methylation density monitoring using quantitative PCR

H. Holemon, Y. Korshunova, J. M. Ordway, J. A. Bedell, R. W. Citek, N. Lakey, J. Leon, M. Finney, John Douglas Mcpherson, J. A. Jeddeloh

Research output: Contribution to journalArticle

43 Scopus citations

Abstract

Aberrant gene silencing of genes through cytosine methylation has been demonstrated during the development of many types of cancers including prostate cancer. Several genes including GSTP1 have been shown to be methylated in prostate cancer leading to the suggestion and demonstration that methylation status of such genes could be used as cancer diagnosis markers alone or in support of histology. We developed a bisulfite-free alternative, MethylScreen technology, an assay for DNA methylation detection utilizing combined restriction from both methylation-sensitive restriction enzymes (MSRE) and methylation-dependent restriction enzymes (MDRE). MethylScreen was used to analyze the 5′ region of GSTP1 in cell lines, in vitro methylated DNA populations, and flash-frozen tissue samples in an effort to characterize the output and analytical performance characteristics of the assay. The output from the quantitative PCR assay suggested that it could not only detect fully methylated molecules in a mixed population below the 1% level, but it could also quantify the abundance of intermediately methylated molecules. Interestingly, the interpreted output from the four quantitative PCRs closely resembled the molecular population as described by clone-based bisulfite genomic sequencing.

Original languageEnglish (US)
Pages (from-to)683-693
Number of pages11
JournalBioTechniques
Volume43
Issue number5
DOIs
StatePublished - Nov 2007
Externally publishedYes

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Biochemistry, Genetics and Molecular Biology(all)

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    Holemon, H., Korshunova, Y., Ordway, J. M., Bedell, J. A., Citek, R. W., Lakey, N., Leon, J., Finney, M., Mcpherson, J. D., & Jeddeloh, J. A. (2007). MethylScreen: DNA methylation density monitoring using quantitative PCR. BioTechniques, 43(5), 683-693. https://doi.org/10.2144/000112597