Assessing high-resolution melt curve analysis for accurate detection of gene variants in complex DNA fragments

Mutation detection has, until recently, relied heavily on the use of gel-based methods that can be both time consuming and difficult to design. Nongel-based systems are therefore important to increase simplicity and improve turn around time without compromising assay sensitivity and accuracy, especially in the diagnostic/clinical setting. In this study, we assessed the latest of the nongel-based methods, namely high-resolution melt (HRM) curve analysis. HRM is a closed-tube method that incorporates a saturating dye during DNA amplification followed by a monitoring of the change in fluorescence as the DNA duplex is denatured by an increasing temperature. We assessed 10 amplicons derived from eight genes, namely SERPINA1, CXCR7, MBL, VDR, NKX3A, NPY, TP53, and HRAS using two platforms, the LightScanner System using LC Green PLUS DNA binding dye (Idaho Technology, Salt Lake City, UT, USA) and the LightCycler 480 using the HRM Master dye (Roche Diagnostics, Indianapolis, IN, USA). DNA variants (mutations or polymorphims) were previously identified using denaturing gradient gel electrophoresis (DGGE) a method, similarly to HRM, based upon the different melting properties of double-stranded DNA. Fragments were selected based on variant and fragment complexity. This included the presence of multiple sequence variants, variants in alternate orientations, and single or multiple variants (constitutional or somatic) in GC-rich fragments. We demonstrate current limitations of the HRM method for the analysis of complex DNA regions and call for caution when using HRM as the sole method to make a clinical diagnosis based on genetic analysis.