Related Experiment Video
Updated: Jun 24, 2026
08:38
Targeted DNA Methylation Analysis by Next-generation Sequencing
Published on: February 24, 2015
Targeted bisulfite sequencing reveals changes in DNA methylation associated with nuclear reprogramming
Jie Deng1, Robert Shoemaker, Bin Xie
1Department of Bioengineering, University of California San Diego, La Jolla, CA, USA.
Nature Biotechnology
|March 31, 2009
Summary
This study introduces a flexible DNA methylation assay for precise digital quantification. The method reveals epigenetic differences between human fibroblasts and pluripotent stem cells, highlighting variations in cytosine methylation.
Area of Science:
- Epigenetics and Genomics
- Molecular Biology
- Stem Cell Biology
Background:
- Current DNA methylation assays lack flexibility and efficiency for large-scale genomic target characterization.
- Understanding epigenetic differences, particularly DNA methylation, is crucial for studying cellular differentiation and reprogramming.
Purpose of the Study:
- To develop and validate a novel method for targeted DNA methylation analysis at single-nucleotide resolution.
- To investigate epigenetic variations associated with cellular dedifferentiation by comparing fibroblasts and pluripotent stem cells.
Main Methods:
- Development of a targeted capture method using ~30,000 padlock probes for specific genomic regions.
- Application of single-molecule bisulfite sequencing for digital quantification of DNA methylation.
- Comparison of DNA methylation patterns in human fibroblast and pluripotent stem cell lines.
Main Results:
- The targeted approach successfully assessed methylation at ~66,000 CpG sites across selected human chromosomes and CpG islands.
- While chromosome-wide methylation patterns were similar, pluripotent cells exhibited slightly higher cytosine methylation than fibroblasts.
- A total of 288 differentially methylated regions were identified between fibroblasts and pluripotent cells, with induced pluripotent stem (iPS) cells showing more methylation than embryonic stem cells.
Conclusions:
- The developed padlock probe-based method offers a flexible and efficient strategy for targeted DNA methylation analysis.
- This approach is highly valuable for analyzing DNA methylation in large and complex genomes, aiding in the study of epigenetic regulation.
- The findings provide insights into epigenetic differences between somatic and pluripotent cells, relevant to stem cell research and regenerative medicine.
Related Concept Videos
01:24Methods of Nuclear Reprogramming
Nuclear reprogramming is a process of transforming one cell type into an unrelated cell type by epigenetic changes that alter the cell’s original gene expression pattern. Such epigenetic changes force cells to express a different set of genes, which play a significant role in inducing transformation into other cell types. Nuclear reprogramming offers applications in reproductive cloning for livestock propagation and regenerative medicine — developing patient-specific cells for injury repair.
01:32Chromatin Modification in iPS Cells
Chromatin modification alters gene expression; therefore, scientists can add histone-modifying enzymes, histone variants, and chromatin remodeling complexes to somatic cells to aid reprogramming into pluripotent stem (iPS) cells.
Compact chromatin makes reprogramming difficult. Enzymes, such as histone demethylases and acetyltransferases, are often added during reprogramming to loosen the chromatin, making the DNA more accessible to transcription factors. Molecules that inhibit histone...
Compact chromatin makes reprogramming difficult. Enzymes, such as histone demethylases and acetyltransferases, are often added during reprogramming to loosen the chromatin, making the DNA more accessible to transcription factors. Molecules that inhibit histone...
01:14Introduction to Nuclear Reprogramming
Nuclear reprogramming is the process of switching gene expression of one cell type to that of another cell type, usually from a differentiated cell state to an undifferentiated cell state. Differentiation occurs during processes such as development and morphogenesis, tissue regeneration, and malignancy. Cells can also be artificially induced to reprogram their gene expression by techniques such as nuclear transfer, induced pluripotency, and cell fusion. Such techniques have many applications in...
01:37Epigenetic Regulation
Epigenetic changes alter the physical structure of the DNA without changing the genetic sequence and often regulate whether genes are turned on or off. This regulation ensures that each cell produces only proteins necessary for its function. For example, proteins that promote bone growth are not produced in muscle cells. Epigenetic mechanisms play an essential role in healthy development. Conversely, precisely regulated epigenetic mechanisms are disrupted in diseases like cancer.
X-chromosome...
X-chromosome...