Epigenetic factors are known to play a critical role in regulating stem cell differentiation by controlling when and how transcription factor bind to promoters or other regulatory elements. Understanding this regulatory mechanism will provide insight into how cells, containing the same genetic material, exhibit significantly different characteristics.
Genetic modifications include the following:
- Genomic imprinting
- X-chromosome inactivation
- DNA methylation
- Histone modifications
These modifications possess unique molecular signatures that are characteristic by cell state (self-renewal vs. differentiated) or cell type (embryonic, early progenitors or lineage specific cells).
DNA Methylation Analysis is used to identify patterns of methylation by relying on bisulfite treatment of methylated genomic DNA to convert unmethylated cytosine to uracil. Methylated cystosine residues remain unchanged. Treated or untreated samples can be directly compared to identify methylated sites and unique patterns of methylation within a site.
The Life Technologies methylSEQr™ Bisulfite Conversion Kit Workflow is based on PCR amplification and direct sequencing to offer greater resolution to identify unique molecular signatures. The methylSEQr™ kit protocol consists of the following:
Experimental Workflow: Find the Products You Need for Every Step
Design high quality PCR primers for methylation mapping experiments using Methyl Primer Express® Software v1.0. Simply cut and paste in your region of interest. The tool searches for CpG islands and simulates bisulfite modification of DNA in silico. Life Technologies supplies all the tools you need to complete methylation mapping experiments.
The methylSEQr™ Bisulfite Conversion Kit converts non-methylated cytosines (C) in your DNA sample to uracil (U). This kit is an initial step in a multi-step workflow that enables you to determine which cytosines in your sample are methylated by comparing the sequences of treated vs. non-treated DNA.
Identify Unique Molecular Signatures using a Streamlined workflow based on BigDye® Terminator v1.1/v3.1 Sequencing Chemistry, BigDye® XTerminator™ Purification Kits, Applied Biosystems Thermal Cyclers and Genetic Analyzers.
- Poster: Cardoso, H.A. and Hart, R.P., Establishing standard assays for quality control and identity traceability of human embryonic stem cells (2007)
- White Paper: Eradication of Cross-Contaminated Cell Lines