Methylation Analysis
MS-HRM Standard Curves With Uniform Resolution From 0% to 100% Methylation.
Methylation Analysis Using Methylation-Sensitive HRM and DNA Sequencing.
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 0.1–2%.
Procedure Overview 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.
Experimental Workflow: Find the Products You Need for Every Step
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Step 1: Bisulfite Conversion of DNA
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.
Products for Step 1: Bisulfite Conversion of DNA
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Cells-to-CpG™ Bisulfite Conversion Kit
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The Cells-to-CpG™ Bisulfite Conversion Kit provides rapid complete conversion of unmethylated cytosine to uracil in reactions that minimize DNA damage and maximize unbiased recovery of DNA.
Step 2: Primer Design
Three main criteria to consider in designing PCR primers are:
- Amplicon length
- The number of potential methylation sites in the amplicon
- Inclusion of CpG dinucleotides in the PCR primer sequence
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.
Products for Step 2: Primer Design
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Methyl Primer Express® Software v1.0
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Offers maximum performance in the minimum time. Fully optimized for Fast cycling, the 7500 Fast delivers high-quality results in as little as 30 minutes.
Step 3: MS-HRM Optimization
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 for the target of interest.
Products for Step 3: MS-HRM Optimization
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ViiA™ 7 Real-Time PCR System
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The new ViiA™ 7 System is a 7th generation real-time PCR system that enables high-productivity qPCR through flexibility, performance, integrated TaqMan® Assay content, and the most advanced features available.
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StepOne™ Real-Time PCR System
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A remarkably simple system designed with a user-friendly, yet powerful, interface for researchers of all experience levels.
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StepOnePlus™ Real-Time PCR System
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A remarkably simple system designed with a user-friendly, yet powerful, interface for researchers of all experience levels.
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7500 Fast Real-Time PCR System
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Offers maximum performance in the minimum time. Fully optimized for Fast cycling, the 7500 Fast delivers high-quality results in as little as 30 minutes.
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7900HT Fast Real-Time PCR System
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The only real-time quantitative PCR system that combines 384-well plate compatibility with fully automated robotic loading—and now also offers optional Fast real-time PCR capability.
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MeltDoctor™ HRM Reagent Kit
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Provides all PCR components and the MeltDoctor™ HRM Dye required for high resolution melting analysis.
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High Resolution Melt (HRM) Software v3.0
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Software enabling high resolution melt curves to be analyzed more accurately for mutation scanning, genotyping, and methylation analysis.
Step 4: MS-HRM Standard Curve & Data Analysis
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.
Products for Step 4: MS-HRM Standard Curve & Data Analysis
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7500 Fast Real-Time PCR System
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Offers maximum performance in the minimum time. Fully optimized for Fast cycling, the 7500 Fast delivers high-quality results in as little as 30 minutes.
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7900HT Fast Real-Time PCR System
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The only real-time quantitative PCR system that combines 384-well plate compatibility with fully automated robotic loading—and now also offers optional Fast real-time PCR capability.
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High Resolution Melt (HRM) Software v3.0
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Software enabling high resolution melt curves to be analyzed more accurately for mutation scanning, genotyping, and methylation analysis.
Step 5: Sequencing the HRM Product
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.
Products for Step 5: Sequencing the HRM Product
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Veriti® 96-Well Fast Thermal Cycler
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Delivers the proven reliability of the 9600 and 9700 GeneAmp® PCR instruments. With the added control of VeriFlex™ Blocks, you have six independent temperature blocks providing precise control over your PCR optimization.
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3130 Genetic Analyzer
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The latest generation of 4-capillary electrophoresis instruments for the low to medium throughput laboratories.
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BigDye XTerminator® Purification Kit
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A fast, simple purification method for DNA sequencing reactions that removes unincorporated BigDye® terminators.
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BigDye® Terminator v3.1 Cycle Sequencing Kits
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Highly flexible chemistry and is ideal for de novo, resequencing, and finishing with PCR product, Plasmid, Fosmid, and BAC templates.
Application Note
Quick Reference Card
Product Bulletin
For research use only. Not intended for any animal or human therapeutic or diagnostic use.
