Methylation Quantification

Epigram

Methylation Quantification at Garvan Molecular Genetics

There is a progressive increase in research on DNA methylation and chromatin related changes in recent years indicating the heightened awareness of its relevance for processes in the regulation of gene expression.

Current studies include the importance of DNA methylation for mammalian development, imprinting and X-chromosome inactivation, suppression of parasitic DNA, and various cancer types. The ability to detect and to quantify methylation is particularly important to the field of cancer diagnostics. Changes in the methylation status of DNA have the potential to serve as an early detection marker for malignancies.

One of our new services uses the Sequenom EpiTYPER system for high throughput quantitative methylation analysis. The EpiTYPER system combines Bisulfite treatment, PCR and a proprietary mass cleavage reaction with MALDI-TOF mass spectrometry analysis to identify CpG site methylation. Sequenom’s EpiTYPER software is used for cleavage pattern analysis. The results can be viewed in what is called an Epigram (pictured above right).

 

How it works

Quantitative Methylation Analysis is a Bisulfite treatment-based method for detection and quantification of DNA methylation. Bisulfite treatment of genomic DNA converts non-methylated cytosine into Uracil while methylated Cytosine remains unchanged. Bisulfite treatment produces methylation-dependent sequence variations of C to T in the amplification products. These C/T variations appear as G/A variations in the cleavage products generated from the reverse strand by base specific cleavage.

These G/A variations result in a mass difference of 16 Da per CpG site, which is easily detected by the MassARRAY system. In the mass spectrum, the relative amount of methylation can be calculated by comparing the signal intensity between the mass signals of methylated and non-methylated template. The method starts with bisulfite treatment of genomic DNA, followed by PCR amplification in which a T7- promoter tag is introduced. The PCR primers should be designed to yield a product within a 200-600 bp range (Bisulfite treatment often limits the success of the PCR when longer amplicons are used).

The significant advantage of this method is that the PCR primers are independent of the methylation state of the genomic DNA, meaning they bind to both methylated and non-methylated template (as opposed to methylation-specific primers). Only two primers are needed to screen for methylation changes within a region of several hundred bases in a single experiment. Then, an in-vitro RNA transcription is performed on the reverse strand, followed by base specific cleavage. In the cleavage reaction, the reverse strand is cleaved by RNase A at specific bases (U or C). Cleavage products are generated for the reverse transcription reactions for both U (T) and C in separate reactions (T is used from this point forward, since it is found in DNA; cleavage, however, actually happens at U in an RNA molecule).

MALDI-TOF MS analyzes the cleavage products, and a distinct signal pair pattern results from the methylated and non-methylated template. Quantitative methylation analysis generates quantitative results for each cleavage product analyzed. Each cleavage product encloses either one CpG site or an aggregate of multiple CpG sites. An analyzed unit containing one or multiple CpG sites is called a “CpG unit.” For both T and C reactions, the resulting cleavage products have the same length and differ only in their nucleotide composition.

Each method has its specific advantages and disadvantages. With the Sequenom system multiple methylated CpG positions in PCR amplified regions of 200-600 bp are analysed. As the software can not differentiate between fragments of the same molecular weight due to overlaps in the peaks for fragments of the same size and cleavage pattern only up to 80% of the CpG sites will be resolved. The huge advantage though is that the detection precision allows for differentiating methylation quantification as low as 5% in sample mixtures. This precision is currently unmatched in any other high throughput analysis method. Other key features are:

  • An alternative to problematic RNA extractions for gene expression determination
  • No need for cloning of PCR products
  • Quantitative assessment of the degree of methylation
  • Simple identification of hypomethylation and hypermethylation
  • Results may be obtained from various sample types, including frozen tissue, mouth swabs, and paraffin embedded tissue
  • High precision, reproducibility and throughput on an established MassARRAY system

Researchers provide reference sequence (up to 600bp per assay) and extracted DNA. PCR oligos are then obtained by Garvan Molecular Genetics and the assay is processed and analysed on the mass spectrometer. The mass spectrometer provides a high level of sensitivity with methylation sites reported to the researcher in the form of a Microsoft Excel spreadsheet.

For further information please also see the guide Introduction to DNA Methylation using the MassARRAY system.

 

Assay design

There are several options for assay designs. There are already established so called Epi-panels which have been demonstrated to work and can be easily applied to your samples. These standard Epi-panels are:

  1. Cancer specific panel (~40 genes)
  2. Human imprinted gene set
  3. Mouse imprinted gene set

For further information please also see Sequenom Standard EpiPanel.

Then there is a library of customer established assays which can be accessed on the Sequenom homepage and contains roughly 400 oncogenes and imprinted regions called the Epi-Browser.

And finally there is the Epi-Designer which allows the design of any gene of your choice but these assays will not be validated.

 

Custom methylation quantification projects at Garvan Molecular Genetics

After you have contacted us for a brief discussion of the type and scope of your project we can give you a quotation and a timeframe for result generation. We will follow a process which has been approved by Sequenom and be in constant communication with you regarding the project progress. We will perform the assay design and order the primers for you, setup PCRs and spot and run the chip(s). We will generate result CSV files which will be emailed to you.

For a discussion of your project and a quote please email us at our .