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April 01, 2009 (Vol. 29, No. 7)

Measurement of Global DNA Methylation

ELISA-Based Method Simplifies Process and Provides Results in HTS Format

  • Epigenetics is often defined as the study of heritable changes that occur without a change in the actual DNA sequences. It has been shown that associated DNA proteins, called histones, are altered by methylation, phosphorylation, acetylation, and other changes. DNA chromatin structure is altered by these histone modifications as well as by direct methylation. These changes are in constant flux and can alter gene expression and related phenotypes; the actual DNA sequence, however, remains unchanged. 

    DNA methylation, the addition of a methyl group to the 5´ carbon of cytosine, is one of the main components of the epigenetic code and is thought to be involved in the repression of gene activity. It’s relationship to development and cancer has been extensively studied. While it is clear that epigenetic regulation is complex, a simplified model starts with double-stranded DNA wound around a set of completely acetylated histones and starting as an activated, fully transcribed gene. 

    Transcriptional repression can be initiated near the promoter by deacetylating specific lysine residues of nearby histone proteins. Subsequently, the lysines can be methylated up to three times per lysine, each time locking in gene shutdown.  Finally, the cytosines within the gene can be methylated at its 5´ carbon to further repress the gene. Although the progression is complex, with some histone methylation actually activating DNA transcription, one may think of this general progression of changes as a compaction of the gene into dense, untranscribable chromatin.

    Recent emphasis has been given to CpG island methylation of the promoter region of specific genes. There are others interested in the overall or global methylation of the entire genome, however. Global methylation varies from diseased to normal states, from tissue to tissue, between genders, and as we age. It is hoped that global methylation profiling can lead to disease-state biomarkers and early diagnosis.

    Several methods exist to measure global methylation levels. Immunohistostaining is commonly used to look at tissue samples, but it has low sensitivity. There are several techniques that use methylation-specific restriction enzymes, but the protocols can be lengthy and cumbersome and often include the use of radioactivity.

    LC-mass spectrometry is considered the gold standard for DNA-methylation analysis as it provides the user with the exact amount of methylated cytosines in a sample. It is costly, however, and requires a certain amount of expertise, and the DNA must be digested to the single nucleotide level prior to analysis. Recently, Sigma Aldrich created a global methylation analysis method, similar to a sandwich ELISA that enables the direct quantitation of genomic DNA methylation. The kit takes four hours, and is amenable to a high-throughput format and friendly to biological researchers.

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