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Targeted DNA Methylation Analysis by Next-generation Sequencing
Published on: February 24, 2015
Screening hypermethylated regions by methylation-sensitive single-strand conformational polymorphism.
H Kinoshita1, Y Shi, C Sandefur
1University of Wisconsin Comprehensive Cancer Center, Department of Surgery, Section of Urology, Department of Environmental Toxicology, University of Wisconsin Medical School, 600 Highland Avenue, Madison, Wisconsin 53792, USA.
Analytical Biochemistry
|February 8, 2000
Summary
Methylation-sensitive SSCP (Ms-SSCP) detects DNA methylation changes in CpG islands. This technique uses bisulfite-treated DNA to identify methylation differences across large DNA regions efficiently.
Area of Science:
- Epigenetics
- Molecular Biology
- Genetics
Background:
- DNA methylation is a key epigenetic modification regulating gene transcription, particularly in CpG islands.
- Detecting methylation patterns is crucial for understanding gene regulation and disease.
- Existing methods for analyzing DNA methylation can be complex or limited in scope.
Purpose of the Study:
- To introduce and validate a novel, sensitive technique for detecting DNA methylation differences.
- To adapt the single-strand conformation polymorphism (SSCP) technique for analyzing methylation status.
- To provide a rapid and accessible method for screening methylation changes in CG-rich regions.
Main Methods:
- The study developed Methylation-sensitive SSCP (Ms-SSCP) by applying SSCP to bisulfite-treated DNA.
- Sodium bisulfite treatment converts unmethylated cytosines to uracil, while 5-methylcytosine remains unchanged.
- PCR amplification with specific primers, followed by nondenaturing gel electrophoresis, reveals methylation-dependent mobility shifts.
Main Results:
- Ms-SSCP effectively detects methylation differences in large (approx. 250 bp) CG-enriched DNA regions.
- The technique is sensitive enough to identify the presence of unmethylated alleles.
- Differential band mobility on gels indicates alterations in DNA methylation density.
Conclusions:
- Ms-SSCP is a rapid, simple, and sensitive method for screening DNA methylation changes.
- It requires only nanogram amounts of DNA and avoids sequencing challenges in CG-rich areas.
- This technique enables the screening of multiple sites across large DNA regions for methylation variations.
