Search Thermo Fisher Scientific
Search Thermo Fisher Scientific
DNA methylation is a key epigenetic mechanism regulating gene expression and chromatin structure.
MS-HRM followed by Sanger-based DNA sequencing is a fast, simple method for methylation analysis that can measure methylation levels as low as 0.1 to 2%.
MS-HRM analysis offers a simple method for quickly analyzing the methylation status of specific genetic loci. Reaction products that merit further analysis can then be sequenced directly to identify precise methylation patterns. Applied Biosystems provides an integrated set of tools for locus-specific DNA methylation analysis using an optimized, streamlined procedure. Figure 1 (Right): MS-HRM Standard Curves With Uniform Resolution From 0% to 100% Methylation. |
The first step of the workflow is to treat genomic DNA with bisulfite, which deaminates unmethylated cytosines (C) to form uracil (U), but does not react with methylated cytosine bases. Thus bisulfite conversion changes the DNA sequence based on the methylation status of individual nucleotides in genomic DNA, and these changes can be detected via HRM analysis.
Cells-to-CpG™ Bisulfite Conversion Kit |
Three main criteria to consider in designing PCR primers are:
These factors significantly influence the sensitivity of the assay; and in some cases can be manipulated to provide discrimination between large or small differences in methylation states.
Methyl Primer Express® Software v1.0 |
In real-time PCR, Ct values should be in the range of 8–30, and fluorescence should increase exponentially through the exponential phase of PCR. MS-HRM melt profiles from 0% and 100% methylated genomic DNA standards should exhibit significant differences.
PCR products run on an agarose gel should indicate that a single product of the expected size is amplified, indicating specificity
MeltDoctor™ HRM Reagents | ||
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High Resolution Melt (HRM) Software |
MS-HRM analysis is based on comparing melt profiles of experimental samples to profiles from DNA with known methylation levels. Universally (or 100%) methylated DNA is commercially available.
As a source of unmethylated DNA, scientists often isolate DNA from blood mononuclear cells.
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High Resolution Melt (HRM) Software |
The final step in methylation analysis is to sequence the amplified PCR products to confirm overall methylation levels and pinpoint the positions of the methylated C residues.
Veriti® 96-Well Fast Thermal Cycler | ||
3130 Genetic Analyzer | ||
BigDye XTerminator® Purification Kit | ||
BigDye® Terminator v3.1 Cycle Sequencing Kits |
The first step of the workflow is to treat genomic DNA with bisulfite, which deaminates unmethylated cytosines (C) to form uracil (U), but does not react with methylated cytosine bases. Thus bisulfite conversion changes the DNA sequence based on the methylation status of individual nucleotides in genomic DNA, and these changes can be detected via HRM analysis.
Cells-to-CpG™ Bisulfite Conversion Kit |
Three main criteria to consider in designing PCR primers are:
These factors significantly influence the sensitivity of the assay; and in some cases can be manipulated to provide discrimination between large or small differences in methylation states.
Methyl Primer Express® Software v1.0 |
In real-time PCR, Ct values should be in the range of 8–30, and fluorescence should increase exponentially through the exponential phase of PCR. MS-HRM melt profiles from 0% and 100% methylated genomic DNA standards should exhibit significant differences.
PCR products run on an agarose gel should indicate that a single product of the expected size is amplified, indicating specificity
MeltDoctor™ HRM Reagents | ||
| ||
High Resolution Melt (HRM) Software |
MS-HRM analysis is based on comparing melt profiles of experimental samples to profiles from DNA with known methylation levels. Universally (or 100%) methylated DNA is commercially available.
As a source of unmethylated DNA, scientists often isolate DNA from blood mononuclear cells.
| ||
High Resolution Melt (HRM) Software |
The final step in methylation analysis is to sequence the amplified PCR products to confirm overall methylation levels and pinpoint the positions of the methylated C residues.
Veriti® 96-Well Fast Thermal Cycler | ||
3130 Genetic Analyzer | ||
BigDye XTerminator® Purification Kit | ||
BigDye® Terminator v3.1 Cycle Sequencing Kits |
Application Note
Methylation Analysis Using Methylation-Sensitive HRM and DNA Sequencing
Quick Reference Card
HRM Methylation Study
Product Bulletin
MeltDoctor™ High Resolution Melt Reagents