Bisulfite Conversion and Other Popular Methods for Measuring Gene-Specific DNA Methylation
Investigate methylation status in specific genes of interest using bisulfite conversion or methylated DNA enrichment methods.
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Bisulfite conversion of cytosine to uracil.
Bisulfite Conversion
To assess methylation status in specific genes of interest, bisulfite conversion of the DNA is often the first step, prior to multiple downstream applications. During bisulfite conversion, DNA is treated with sodium bisulfite, which converts the unmethylated cytosines to uracils, whereas the methylated cytosines are unsusceptible to treatment and remain as cytosines. During amplification of the DNA, the uracils are then read as thymines, while the methylated cytosines remain as cytosines. This allows for discrimination of the methylation status of the cytosines.
Bisulfite DNA conversion combined with sequencing (bisulfite-sequencing), is the gold standard for DNA methylation studies, as it provides gene-specific base-pair resolution information on the methylation status of each individual cytosine. During sequencing, the unmethylated cytosines will be read as thymines, whereas the methylated are read as cytosines, allowing to discriminate the methylation status of the cytosines. Bisulfite-sequencing can be performed at a whole-genome scale or in a more targeted gene-specific manner.
Constructing Illumina-compatible libraries directly from bisulfite-treated DNA for Methyl-Seq can be accomplished with the EpiNext Post-Bisulfite DNA Library Preparation Kit (Illumina). Prepare a DNA library -- after successful bisulfite conversion -- for various Illumina platform-based bisulfite sequencing (bisulfite-seq) assays, such as whole genome bisulfite sequencing (WGBS), oxidative bisulfite sequencing (oxBs-seq), reduced representation bisulfite sequencing (RRBS), and other bisulfite-based next generation sequencing applications.
For streamlined bisulfite sequencing NGS library preparation from low input DNA, the EpiNext High-Sensitivity Bisulfite-Seq Kit (Illumina) can be used. This kit is designed to bisulfite-convert DNA and prepare an Illumina-based library for bisulfite sequencing, all in one kit.
Bisulfite DNA conversion combined with quantitative PCR (Methylation Specific-PCR) allows gene-specific assessment of the methylation status of a specific region of DNA. However it does not provide information on each individual cytosine site in the region being interrogated. Often times, it represents a powerful technique to validate the methylation results obtained by bisulfite sequencing, and a less manually laborious and cost-effective technique to assess the methylation status of genes of interest in many samples simultaneously. Perform fast, specific, and sensitive methylation-specific quantitative PCR with EpiGentek's Methylamp MS-qPCR Fast Kit
Enrichment of methylated DNA (meDIP or MBD)
Affinity-based methods for enrichment of the methylated DNA, such as methylated DNA immunoprecipitation (meDIP) or Methyl-CpG binding domain (MBD), are alternative techniques that don’t rely on bisulfite treatment of DNA.
Methylated DNA Immunoprecipitation (meDIP)
The methylated status of specific genes or regions in the genome can be assessed by enrichment of methylated DNA by immunoprecipitation using an antibody specific for 5-mC (meDIP). This technique does not require modification of DNA by bisulfite treatment. The methylated DNA obtained can then be sequenced by NGS (meDIP-Seq) or analyzed by qPCR (meDIP-PCR). This technique can also be used for immunoprecipitation of 5-hmC, in contrast to bisulfite-sequencing, which cannot discriminate between 5-mC and 5-hmC.
The methylated DNA can also be enriched by pull down using MBD protein. MBD proteins bind methylated DNA, preferentially at CpG dinucleotides, and mediate gene silencing by recruiting additional protein complexes. EpiGentek offers two kits, for use in cell lines and tissues, for chromatin immunoprecipitation of the methylated DNA, based on MBD pull down using an antibody specific for MBD2.
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