MethylDetect’s goal is to make the MS-HRM technology easy accessible both for new-comers and experienced researchers in the field of epigenetics.
We offer kits for DNA methylation detection that are ready-to-use and compatible with standard laboratory equipment


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Epigenetics is a study of the heritable gene expression changes that do not involve alternations in DNA sequence. Prefix epi- signifies “on top of” or “in addition to”, hence epi-genetic is understood as something on top of genetics that changes the expression of the genes.

The “epi- mechanisms” that influence gene expression are most frequently classified as:

  • Covalent modification of the DNA bases. DNA methylation is the most studied DNA modification leading to gene expression changes (see also: What is DNA methylation?).
  • Histone modifications. DNA in the nucleus does not exist by itself but in a complex with proteins called histones. Those proteins can regulate access of the transcription machinery to the DNA and hence influence gene expression. This type of gene expression regulation is accomplished by covalent modifications of the histones.
  • Regulation of gene expression by non-coding RNA mechanisms such as micro-RNA or siRNA.

Enzymatic addition of methyl groups (CH3-) to the DNA molecule is referred to as DNA methylation. In mammals the methyl groups are almost exclusively added to cytosines at CpG dinucleotides (Figure 1). Specific genomic regions have a high density of CpG dinucleotides and are defined as CpG islands (CGI). When CGIs are located in promoter regions DNA methylation typically acts to repress gene transcription (Figure 2).

Over 60% of human protein coding genes contains a CGI in the promoter and can potentially be regulated by DNA methylation. DNA methylation regulates processes, such as imprinting, X-chromosome inactivation, transcriptional repression of transposable elements and aging.

Abnormal changes in DNA methylation have been identified in many different diseases as cancer, inflammatory, autoimmune, psychiatric, cardiovascular, and age-related diseases

Figure 1:

Firgure 2:

During DNA replication (cell division) methyl groups (DNA methylation) are resynthesized on the newly replicated strand by one of the enzymes called DNA Methyl Transferases (DNMT). Those enzymes are not present in standard PCR reaction. Therefore, a PCR product obtained from amplification of a specific locus does not contain information about methylation status of the cytosines and the methylation information is lost.

To analyze the methylation pattern of cytosines within the locus of interest the information about which cytosines are methylated needs to be preserved before PCR amplification is performed. Sodium bisulfite deaminates non-methylated cytosines to uracil and leaves methylated cytosines untouched (in other words methylated cytosines are resistant to modification induced by sodium bisulfite).

Thus, after bisulfite treatment the cytosine content of the template depends on the number of methylated cytosines in the untreated template (protected from change) as shown below in part B and C of the diagram.

PCR products with different number of cytosines can easily be distinguished in post PCR analyses such as Methylation Sensitive High-Resolution Melting (MS-HRM) (see: How does MS-HRM work?).

A: Genomic DNA template (in bold CG sites, which are the dinucleotides undergoing methylation in humans):

Figure 1:

After bisulfite modification:

B: Template with methylated cytosines at CG sites – only Cs not protected by methyl groups are changed by sodium bisulfite:

Figure 2:

C: Template with non-methylated cytosines at CG sites – all the Cs are changed by sodium bisulfite:

Figure 3:

Dissociation of the double stranded DNA helix into single coils is referred to as DNA melting. It can be accomplished by simply heating double stranded DNA. The temperature at which the DNA strands dissociates into single coils depends on the number of hydrogen bonds holding the complementary strands. There are two hydrogen bonds between adenine (A) and thymine (T) and three bonds between guanine (G) and cytosine (C). Therefore, in principle the more G and C in the sequence the higher temperature is needed to melt a given DNA fragment.

How to detect difference in melting temperature of PCR products?

The most commonly used method to determine the melting temperature of a PCR product is to subject the product to a temperature gradient in the presence of intercalating dye. The intercalating dyes are chemicals that only emit light when bound to double stranded DNA. In a typical melting experiment, a PCR product is mixed with an intercalating dye, and fluorescence emitted by this mix is monitored as the sample is slowly heated (subjected to a temperature gradient).

The outcome of the analysis is a curve displaying fluorescence changes emitted by the sample over the range of temperature that the sample was subjected to, commonly referred to as a melting profile (Figure 1).  At the beginning of the melting experiment the temperature is low and all PCR product in the sample is double stranded. Thus, we observe high levels of florescence from the sample (Figure 1 – A). We continue to observe high levels of fluorescence, as the temperature increases up to the point, where all hydrogen bonds within the PCR fragment are broken and the amount of double stranded PCR product drastically decreases. Consequently, we observe a sharp decrease in the detected fluorescence level (Figure 1 – B). At a high temperature there is no double stranded PCR product in the sample and the fluorescence levels are close to 0 (Figure 1 – C). The temperature at which we observe the sharp drop in the fluorescence depends on the number of hydrogen bands in the analyzed PCR product and hence is specific to analyzed fragment.

This type of analysis can concurrently be performed on standard RT- qPCR instrument as a majority of those instruments have built in modules that allow post-PCR melting analyses.

Figure 1:

Methylation-Sensitive High-Resolution Melting (MS-HRM) is based on a PCR methylation detection method.

The number of cytosines (C) in a PCR product, amplified from a sodium bisulfite modified template, depends on the methylation status of cytosines in the un-modified template. Sodium bisulfite preserves the methylated cytosines and modifies non-methylated cytosines into uracil, which subsequently is substituted with thymine during PCR. Thus, if the locus is methylated, a C rich PCR product is obtained after PCR amplification (see A below), and a T rich PCR product if the locus was non-methylated (see B below). These PCR products will display different melting profiles (see also: What is DNA melting?) when subjected to Methylation-Sensitive High-Resolution Melting (MS-HRM) analyses.

The PCR product amplified from the non-methylated version of a specific locus will have relatively low melting temperature and melts earlier in the temperature gradient (Figure 1 – red curve) than the PCR product amplified from the methylated version of the same locus, which will melt at relatively higher temperature (Figure 1 – blue curve).

In summary, MS-HRM is a PCR methylation detection method, dependant on bisulfite modification, and enables cost effective, fast, and highly sensitive assessment of locus specific methylation status.

A: PCR product obtained from the methylated version of the locus:

B: PCR product obtained from the non-methylated version of the locus:

Figure 1:

Further reading:

  1. MS-HRM protocol in Nature Protocols.

PMID:19180074

  1. MS-HRM in Nucleic Acid Research

PMID:17289753

  1. MS-HRM protocol in Methods in Molecular Biology

PMID:18987818

PMID:29224163

The methylated and non-methylated locus after bisulfite modification differs in number of cytosines (for detail see: What is bisulfite modification?). Different base composition of the template may lead to the differences in the efficiency of PCR amplification of those two versions of a locus and this phenomenon is referred to as PCR bias. PCR bias in methylation studies was first identified by Warnecke PM et al. PMID:9336479. This phenomenon was shown to present a serious pitfall in PCR based methylation detection leading to underestimation of methylation content of the screened sample.

PCR bias presents a serious challenge especially in experiments that aim to detect methylated alleles in a sample where it is present at low frequency in a background of non-methylated alleles. In those samples higher efficiency of PCR amplification of the non-methylated allele may even lead to lack of ability to detect the methylated allele and consequently false negative result, described in: PMID:19483476.

Sample material with low content of methylated alleles of interest and a high background of non-methylated allele are for example:

  • whole blood where only a specific type of cells may contain the methylated allele of interest and the vast majority of the cells contain non-methylated alleles.
  • FFPE samples where the methylated allele in the DNA sample is underrepresented due to significant DNA degradation.
  • liquid biopsies where only a few copies of the methylated allele may be present due to the nature of the sample.

The innovative primer design for Methylation-Sensitive High-Resolution Meting technology (MS-HRM) allows to overcome PCR bias and enables highly sensitive methylation detection even in samples where the methylated allele is significantly underrepresented.

For details of primer design see: PMID:18710507

Following good laboratory practice any experiment needs to be properly controlled. In PCR based methylation screening experiments two controls are necessary: methylated (positive) and non-methylated (negative). Those controls are normally chemically modified DNA templates consisting of methylated and non-methylated versions of the locus of interest and are reference points for the analyses of an unknown sample.

In the Methylation-Sensitive High-Resolution Melting (MS-HRM) protocol methylation status of a screened sample is assessed by comparison of the melting profile (see also: What is DNA melting?)  of the PCR product amplified from a screened sample (Figure 1 – green) with the melting profiles of PCR products amplified from methylated (Figure 1 – blue) and non-methylated (Figure 1 – red) controls.

In principle, hypomethylation is observed if the melting profile of a screened sample does not overlap with the methylation profile of the positive control and hypermethylation in observed when the melting profile of the sample does not overlap with the melting profile of a non-methylated control.

Figure 1:

Formalin-Fixation followed by paraffin-embedment (FFPE) is one of the most widely practiced methods for preservation and archiving of clinical tissue samples. It is estimated that worldwide, over a billion FFPE tissue samples are being stored in numerous hospitals, tissue banks, and research laboratories. These archived samples could potentially provide a wealth of information in retrospective molecular studies (from: PMID:20147068 ). The retrospective molecular studies based on well-described clinical material are essential for validation of the clinical utility of molecular biomarkers including methylation-based biomarkers.

The Methylation-Sensitive High-Resolution melting technology (MS-HRM) was shown to allow for stable methylation assessment in 30 years old archival FFPE material, see: PMID:20025721. Moreover, the level of degradation of DNA in FFPE samples was shown not to affect the performance of methylation detection by Methylation-Sensitive High-Resolution Melting (MS-HRM), see: PMID:26551081.

Tumor DNA is secreted into body fluids that come in direct contact with the tumor site. Body fluids such as urine, plasma or sputum containing DNA from the tumor site is referred to as a liquid biopsy. Due to its nature liquid biopsies can be obtained in non-invasive or almost non-invasive manner, which makes them a very attractive sample source for molecular diagnostics, see: PMID:29872715.

DNA methylation biomarkers have been shown to be readily detectable in liquid biopsies. Moreover, tests for early cancer detection or relapse monitoring are already in use in clinical disease management.

Methylation-Sensitive High-Resolution Melting (MS-HRM) is a technology that can be utilized for methylation biomarkers detection in liquid biopsies.

Product Panels

Apoptosis DAPK1 TNFRSF25(DR3) TNFRSF21­
Breast Cancer BRCA1 BRCA2 TIMP3
Colon cancer ZNF442 WRN VIM
Gastric Cancer TIMP3 GSTP1 DAPK1
Leukemia and Lymphoma GSTP1 DAPK1 LATS2
Liver Cancer TIMP3 GSTP1 DAPK1
Lung Cancer GSTP1 DAPK1 ONECUT2
Melanoma TIMP3 DAPK1 CCND2
Prostate Cancer TIMP3 GSTP1 PITX2
Cell cycle TP53 RBL1 RAD9A
Cytokine Production TRAF2 TRAF6 TLR2
DNA Methylation Assay P16 SEPT9 TIMP3
DNA Repair MLH1 BRCA1 BRCA2
Homeobox Genes (HOX) LHX1 PITX2 SIX6
Inflammatory response and autoimmunity ABCF1 TYK2 TRAF2
Mental Disorders TERT TFAM SORBS3
Notch Signaling Pathway SNW1 RBPJL PSENEN
Polycomb Genes PcG TRIM27 SUZ12 RING1
Stem Cell Transcription Factors EGFR PITX2 WT1
Stress response and Toxicity XRCC6(G22P1) XBP1 XRCC2
T Helper Cell Differentiation TNFSF11 TGIF1 TBX21
T-Cell and B-Cell Activation THY1 SOCS1 NCK1
TGFbBMP Signaling Pathway TGFB1 TGFB2 TGFB3
TollLike Receptor TLR Signaling UBE2V1 TRAF6 TOLLIP
Tumor Suppressor Genes TSG APC MGMT RASSF1A
WNT Signaling WNT10A WNT10B WNT5A

DNA Methylation Testing Kits

DNA Methylation is one of the most widely studied epigenetic mechanisms of gene expression regulation. Aberrant methylation of increasing number of genes is being shown to contribute to the majority of diseases such as cancer, cardiovascular disorders, aging, obesity, allergy, diabetes, depression, Alzheimer’s, Parkinson’s or schizophrenia.
Solid research evidence already shows that DNA methylation-based biomarkers can be utilized at all stages of clinical disease management including:

  • Disease risk assessment
  • Early detection
  • Personalization of treatment
  • Monitoring of chronic disorders and recurrence

The technology was already used for the detection of methylation biomarkers in fields like forensics and such a difficult DNA material sources as liquid biopsies or FFPE tissues.
MethylDetect offers ready-to-use kits for detection of aberrant gene methylation. The kits are based on the MS-HRM (Methylation Sensitive High Resolution Melting) technology and can be used with standard laboratory equipment. Additionally, our new controls system shipped with each kit, provides state-of-the-art experimental reliability.

Methylation detection with MethylDetect’s Kit

1. ADD: The MethylDetect kit components to the bisulfite-modified biological sample and PCR reagents
2. AMPLIFY: Use standard laboratory Real Time-PCR system to PCR amplify the target
3. DETECT: Assess the methylation status of the sequence of interest with a High-Resolution Melting module

Principles of methylation detection with MethyDetect Kit