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.

Recommended Products

 

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

  • QuantStudio™ 1
  • QuantStudio™ 3
  • QuantStudio™ 5
  • 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.

 

 

High Resolution Melt (HRM) Software
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

 

Real-Time PCR Instruments

  • QuantStudio™ 1
  • QuantStudio™ 3
  • QuantStudio™ 5
  • 7900HT Fast Real-Time PCR System: a real-time quantitative PCR system that combines 384-well plate compatibility with fully automated robotic loading as well as optional Fast real-time PCR capability.
  • ViiA™ 7 Real-Time PCR System: enables high-productivity qPCR through ultimate flexibility, performance, and the most advanced features available.

 

 

High Resolution Melt (HRM) Software
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.

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.

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® 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. 

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.

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.

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.

Recommended Products

 

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.

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

Recommended Products

 

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

 

Real-Time PCR Instruments

  • QuantStudio™ 1
  • QuantStudio™ 3
  • QuantStudio™ 5
  • 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.

 

 

High Resolution Melt (HRM) Software
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.

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.

Recommended Products

 

Real-Time PCR Instruments

  • QuantStudio™ 1
  • QuantStudio™ 3
  • QuantStudio™ 5
  • 7900HT Fast Real-Time PCR System: a real-time quantitative PCR system that combines 384-well plate compatibility with fully automated robotic loading as well as optional Fast real-time PCR capability.
  • ViiA™ 7 Real-Time PCR System: enables high-productivity qPCR through ultimate flexibility, performance, and the most advanced features available.

 

 

High Resolution Melt (HRM) Software
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.

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.

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® 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. 

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