Methylation Analysis | Life Technologies

Methylation Analysis

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 to 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.

Figure 1 (Right): MS-HRM Standard Curves With Uniform Resolution From 0% to 100% Methylation.

Methylation Analysis Experimental Workflow

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.

Recommended Products

Cells-to-CpG™ Bisulfite Conversion Kit
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.

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.

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Methyl Primer Express® Software v1.0
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.

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

Recommended Products

MeltDoctor™ HRM Reagents
Provides all PCR components and the MeltDoctor™ HRM Dye required for high resolution melting analysis.

Real-Time PCR Instruments

ViiA™ 7 Real-Time PCR System: 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.
StepOne™ Real-Time PCR System: A remarkably simple system designed with a user-friendly, yet powerful, interface for researchers of all experience levels.
StepOnePlus™ Real-Time PCR System: A remarkably simple system designed with a user-friendly, yet powerful, interface for researchers of all experience levels.
7500 Fast Real-Time PCR System: 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.
7900HT Fast Real-Time PCR System: 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.

High Resolution Melt (HRM) Software v3.0
High Resolution Melting (HRM) analysis is an alternative to dHPLC sequencing screening of new gene variants. The application does not require temperature shifting, which results in a greater likelihood of identifying new homozygous mutations than methods that require temperature shifting.

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.

Recommended Products

		Veriti® 96-Well Fast Thermal Cycler 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.
		3130 Genetic Analyzer The latest generation of 4-capillary electrophoresis instruments for the low to medium throughput laboratories.
		BigDye XTerminator® Purification Kit A fast, simple purification method for DNA sequencing reactions that removes unincorporated BigDye® terminators. BigDye XTerminator® Purification Kit - 50 mL (~2,500 - 20 µL reactions) BigDye XTerminator® Purification Kit - 800 mL BigDye XTerminator® Purification Kit - 2 mL (~100 - 20 µL reactions) BigDye XTerminator® Purification Kit - 20 mL (~1,000 - 20 µL reactions)
		BigDye® Terminator v3.1 Cycle Sequencing Kits Highly flexible chemistry and is ideal for de novo, resequencing, and finishing with PCR product, Plasmid, Fosmid, and BAC templates. BigDye® Terminator v3.1 Cycle Sequencing Kit, 24 rxn BigDye® Terminator v3.1 Cycle Sequencing Kit, 100 rxn BigDye® Terminator v3.1 Cycle Sequencing Kit, 1000 rxn BigDye® Terminator v3.1 Cycle Sequencing Kit, 5000 rxn BigDye® Terminator v3.1 Cycle Sequencing Kit, 25000 rxn

Literature

Application Note
Methylation Analysis Using Methylation-Sensitive HRM and DNA Sequencing

Quick Reference Card
HRM Methylation Study

Product Bulletin
MeltDoctor™ High Resolution Melt Reagents