Abstract
BackgroundGenomic DNA methylation affects approximately 1% of DNA bases in humans, with the most common event being the addition of a methyl group to the cytosine residue present in the CpG (cytosine-guanine) dinucleotide. Methylation is of particular interest because of its role in gene silencing in many pathological conditions. CpG methylation can be measured using a wide range of techniques, including methylation-specific (MS) PCR, pyrosequencing (PSQ), bisulfite sequencing (BS) and methylation-sensitive restriction enzyme (MSRE) PCR. However, although it is possible to utilise these methods to measure CpG methylation, optimisation of the assays can be complicated due to the absence of suitable control DNA samples.ResultsTo address this problem, we have developed an approach that employs multiple displacement based whole genome amplification (WGA) with or without SssI-methylase treatment to generate CpG methylated and CpG unmethylated DNA, respectively, that come from the same source DNA.ConclusionUsing these alternately methylated DNA samples, we have been able to develop and optimise reliable MS-PCR, PSQ, BS and MRSE-PCR assays for CpG methylation detection, which would otherwise not have been possible, or at least have been significantly more difficult.
Highlights
Genomic DNA methylation affects approximately 1% of DNA bases in humans, with the most common event being the addition of a methyl group to the cytosine residue present in the CpG dinucleotide
The analysis of MMP-2 methylation status indicated that both MDAMB231 and multiple displacement amplification (MDA)-MB468 were methylated, whilst HFFF2 was unmethylated (Figure 2b)
When HFFF2, MDA-MB231 and MDA-MB468 cell line DNAs were subjected to HpyCH4IV digestion and MMP-1 and MMP-3 PCR the results demonstrated that the CpG site in the MMP-1 amplicon was methylated in HFFF2 and MDA-MB468, but unmethylated in MDA-MB231 (Figure 4b)
Summary
Genomic DNA methylation affects approximately 1% of DNA bases in humans, with the most common event being the addition of a methyl group to the cytosine residue present in the CpG (cytosine-guanine) dinucleotide. Promoter methylation has an important role in controlling the binding of transcription factors and other proteins to the DNA, which in turn modulate the association of methyl-DNAbinding proteins and histone deacetylases to the transcription start sites. This modulation is critical in regulating the switch between transcriptionally active euchromatin (unmethylated) and transcriptionally silent heterochromatin (methylated) and in turn gene expression [1,2]. The most common methylation event is the addition of a methyl group to the cytosine present in the CpG (cytosine-guanine) dinucleotide [3] These dinucleotides exist as either CpG islands or as sparsely distributed CpG motifs within the promoter regions of many genes. Hypermethylation (methylation) of these islands or motifs results in transcriptional silencing [4], whilst hypomethylation (demethylation), either global or gene specific, induces expression [5]
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