Methylation-sensitive high-resolution melting in the context of legislative requirements for validation of analytical procedures for diagnostic applications

Abstract

Temperature gradient was first used to identify the methylation status of the DNA sequence over 10 years ago; however, the initially published protocol was shown to have poor analytical sensitivity. Recent developments in the field of DNA melting technologies, combined with the identification of procedures to overcome the sensitivity issues in the PCR-based methylation detection applications, led to the development of the methylation-sensitive high-resolution melting (MS-HRM) protocol. This protocol allows for highly sensitive detection of methylation levels in a labor- and cost-efficient fashion. Moreover, it enables investigation of methylation status of imprinted loci as well as identification of heterogeneous methylation. The MS-HRM technology is being increasingly applied in research laboratories and has a potential for future application in diagnostic settings. The focus of this article is to describe the development of the HRM technology for methylation analyses and evaluate the diagnostic applicability of the MS-HRM technology.

Keywords::

• biomarker
• cancer
• diagnostics
• methylation
• MS-HRM

Acknowledgements

TK Wojdacz would like to thank Dr Lise Lotte Hansen for the critical review of this manuscript.

Financial & competing interests disclosure

TK Wojdacz is listed as an inventor on the patent pending application on the aspects of MS-HRM technology. TK Wojdacz would like to thank The Danish Cancer Society, CIRRO – The Lundbeck Foundation Center for Interventional Research in Radiation Oncology and The Danish Council for Strategic Research for the support of this work. The author has no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.