Introduction

High-Resolution Melting (HRM) analysis is a relatively new, post-PCR analysis method used to identify variations in nucleic acid sequences. The method is based on detecting small differences in PCR melting (dissociation) curves. It is enabled by improved dsDNA-binding dyes used in conjunction with real-time PCR instrumentation that has precise temperature ramp control and advanced data capture capabilities. Data are analyzed and manipulated using software designed specifically for HRM analysis

Advantages of HRM

  • Low reagent consumption, with little waste: HRM requires only PCR reaction volume (20 µL) for analysis of each sample, eliminating the need for HPLC solvents or DGGE gels
  • Simple, fast workflow: no additional instrumentation is required after PCR amplification. A high-resolution melt curve can be added to the end of the amplification run and analyzed immediately after completion
  • Fast optimization: unlike DHPLC, thermal optimization is not required
  • Low sample consumption: following HRM analysis, the PCR amplicon can be used directly in a Sanger sequencing reaction
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