Methylation-specific PCR (MSP)
Gene-specific, base resolution DNA methylation
A slightly more advanced method for beginners looking to start in DNA methylation research is gene-specific, base-resolution DNA methylation analysis. This assesses the amount of DNA methylation of a specific gene at base resolution, compared to global DNA methylation, which provides an overall view of the entire methylation status of the genome. The BisulFlash DNA Modification Kit is the ideal kit for quick bisulfite treatment of DNA geared towards methylation-specific PCR analysis. MSP begins with bisulfite conversion wherein unmethylated cytosine is converted to uracil. In subsequent PCR amplification, uracil is recognized as thymine, leaving the methylated cytosines (5-mC) unaffected. This allows the amount of methylation to be measured. Protecting against DNA degradation caused by the typically harsh chemical reaction is vital in order to ensure optimal recovery and accurate methylation status.
Use these methylation-specific PCR resources to enhance your epigenetic study:
DNA Bisulfite Sequencing (Methyl-Seq)
Genome-wide, base resolution DNA methylation
For those just beginning epigenetic research, next-generation sequencing (NGS) may seem daunting. There is typically a lot of data produced and significant time must be devoted to interpretation and figure generation. Massively-parallel sequencing platforms, such as Illumina or PacBio, have become invaluable tools that revolutionized genome and epigenome analysis. NGS gives researchers a deeper and far more detailed look into their samples than ever before. NGS technology delivers robust, unprecedentedly high-quality data.
Common DNA methylation-based applications include whole genome (WGBS), reduced representation (RRBS), and targeted bisulfite-seq. Successful bisulfite sequencing projects start with complete and efficient bisulfite conversion. Samples can be sequenced on an Illumina instrument for precise identification of methylated genes of the sample. The EpiNext High-Sensitivity Bisulfite-Seq Kit (Illumina) is ideal for streamlined bisulfite sequencing NGS library preparation from low input DNA. Because it’s an all-in-one kit, it includes everything needed for bisulfite conversion and library preparation and is excellent for beginners.
Investigating histone modifications is another route to pursue for those just getting started in epigenetic research. Post-translational modifications (PTMs) which occur to histones are thought to play a crucial role in transcription repression or activation. Such histone modifications include histone methylation, histone acetylation, and histone phosphorylation. These epigenetic mechanisms and the way they impact chromatin structure or recruit histone modifiers brings us closer to understanding the factors that alter gene expression and the consequences of these changes.
Histone modifications can impact the structure by loosening or tightening chromatin. Looser chromatin is known as euchromatin, which is more accessible to transcription and increases gene expression. Heterochromatin, on the other hand, is more tightly compacted, making it less accessible to transcription and, as a result, reducing gene expression. New insights into different histone modifications and their effect on cellular processes begin to unveil what some refer to as the “histone code.”
Global H3 or H4 Modification ELISA
Total quantification of well-characterized H3 or H4 modifications
Similar to beginning in global DNA methylation research, when getting started in histone modifications it is very useful to have an overview of your samples. This will save time and effort in the future and can help narrow down areas for further experimentation.
Investigate 21 different histone H3 modifications or 10 different histone H4 modifications on one microplate using the EpiQuik Histone H3 Modification Multiplex Assay Kit or the EpiQuik Histone H4 Modification Multiplex Assay Kit. This high throughput method will accurately quantify amounts of identified histone proteins at specific H3 or H4 modification sites present in your sample prior to getting involved in more advanced downstream applications such as ChIP-sequencing (ChIP-seq).
If you have a single histone methylation or histone acetylation modification you’d like to measure, use these histone methylation quantification and histone acetylation quantification kits. Popular kits include:
Gene-specific identification of histone modifications
Chromatin immunoprecipitation, or ChIP, is a popular epigenetic research method for investigating protein-DNA binding interactions. This method can be used for identifying histone modifications at the gene-specific level. Chromatin immunoprecipitation involves the binding of proteins to particular DNA sequences, allowing researchers to investigate transcription factors that interact with target genes and histone modifications occurring at genomic locations. Briefly, it involves crosslinking proteins to DNA, shearing the chromatin via sonication, and immunoprecipitating with a specific antibody. Then, the protein-DNA complexes are reverse crosslinked so the DNA fragments can be analyzed. Various downstream applications of ChIP include ChIP-sequencing, ChIP-PCR, and ChIP-on-chip (microarrays). The ChromaFlash High-Sensitivity ChIP Kit is optimal for immunoprecipitation of chromatin (ChIP) from small amounts of mammalian cells or tissues.
For an in-depth technical guide to ChIP, see A Starter Chromatin Immunoprecipitation (ChIP) Protocol at What is Epigenetics? or read the three-part series A Technical Guide to Conquering ChIP by David Esopi, research specialist at Johns Hopkins Sidney Kimmel Comprehensive Cancer Center.
Genome-wide mapping and identification of histone modifications.
This method combines chromatin immunoprecipitation (ChIP) with massively parallel sequencing technology to precisely analyze protein interactions with DNA. ChIP-Seq is used for pinpointing the exact gene location to which the protein of interest binds, but like bisulfite sequencing, produces significant amounts of data. Use the EpiNext ChIP-Seq High-Sensitivity Kit (Illumina) Kit to perform ChIP and library preparation in a single kit.
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