HRM - HIGH RESOLUTION MELTING

High Resolution Melting

The High Resolution Melting (HRM) analysis method is used for identifying genetic variation in nucleic acid sequences.
HRM is based on PCR melting (dissociation) curve techniques.


HRM can be used to discriminate DNA sequences based on their composition, length, GC content, methylation status or strand complementarity.

EpiMelt MS-HRM kit is designed for optimal performance of methylation detection.

EpiMelt MS-HRM kit

EpiMelt MS-HRM kit is designed for optimal performance of methylation detection.

DNA methylation changes are involved in the development of many diseases including cancer, heart diseases, diabetes and mental illnesses.  Furthermore, the MS-HRM technology is used for detection of DNA methylation biomarkers in fields like forensics, and for investigating challenging DNA materiel sources as liquid biopsies and FFPE tissues.


F
EATURES:

  • Detection of heterogeneous methylation status
  • Choose between 850 specific gene-specific loci
  • Fast results
  • User-friendly
  • High sensitivity
  • High reproducibility
  • Less sample required

DESCRIPTION

EpiMelt MS-HRM kit consists of the EpiMelt MS-HRM 2x Master Mix and the DNA Methylation Assay Controls (gene specific primers + methylation controls).  The kit is available in 3 sizes: 200, 500 and 2500 reactions.

The EpiMelt MS-HRM 2x Master Mix is specifically developed by Ampliqon A/S for optimal performance with EpiMelt Assay Controls, which are developed and manufactured by MethylDetect ApS. MethylDetect offers more than 850 different ready-to-use kits for detection of aberrant gene methylation. The kit solution is based on the MS-HRM (Methylation Sensitive High Resolution Melting) technology. The control system shipped with each kit, provides state-of-the-art experimental reliability.

THE EPIMELT MS-HRM METHOD – DETECTION OF METHYLATION CHANGES

1. Bisulfite modification of DNA template

2. PCR amplification of bisulfite modified DNA template + controls

3. Methylation-Sensitive (MS) High-Resolution Melting (HRM) analysis

1. BISULFITE MODIFICATION

To analyse 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). Therefore, after bisulfite treatment the cytosine content of the template depends on the number of methylated cytosines in the untreated template. More information about bisulfite modifications.

2. PCR AMPLIFICATION

The number of cytosines (C) in the PCR product amplified from a bisulfite modified template depends on methylation status of the DNA template before bisulfite modification. Highly methylated DNA  results in GC-rich PCR products, whereas AT-rich PCR products are obtained from non-methylated DNA.

Gene specific primers and three controls are included in the EpiMelt MS-HRM kit: a positive control (100 % methylated), Assay Calibration control (specified % of methylation) and a negative control (non-methylated) DNA template of the specific gene and serve as reference points for the analyses of an unknown sample.

Amplification of the specific locus and the three assay controls is performed using the EpiMelt MS-HRM 2x Master Mix.

PCR set up, when using the EpiMelt MS-HRM kit. Samples and controls are analysed in triplets.

3. MS-HRM

The obtained PCR products display different melting profiles when subjected to MS-HRM analyses.

MS-HRM results: Methylation status of the sample is assessed by comparison of the melting profile of the PCR product amplified from the sample (green) with the melting profiles of PCR products amplified from methylated (blue) and non-methylated (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 hyper-methylation in observed when the melting profile of the sample does not overlap with the melting profile of a non-methylated control.

Assessing the methylation status of the gene sequence of interest with a High-Resolution Melting module