Real-Time PCR High-Resolution Melting Analysis

High Resolution Melting (HRM) is a homogeneous, exceptionally incredible innovation for single nucleotide polymorphism (SNP) genotyping, mutation scanning and sequence scanning in DNA samples. HRM analysis works on the principle of melting (dissociation) curve methodologies of Polymerase Chain Reaction (PCR) empowered by the new accessibility of improved double-stranded DNA (dsDNA)–binding dyes and next-generation real-time PCR instrumentation. The HRM technology portrays samples of nucleic acids on the basis of their disassociation behaviors and identifies the differences in even the short sequence in amplified PCR products, just by direct melting. Samples are further distinguished according to the length of their sequence, GC content and strand complementarity. Indeed, even a single change in the base pair in the sequences of DNA samples causes differences in the HRM curve. The difference in the melting curves of different genetic sequences at distinct rates can be observed, detected and compared using these curves. Development of the melting curves after HRM analysis is basically plotted with temperature on the X axis and fluorescence on the Y axis, which resembles the real-time PCR amplification curve but with the difference of temperature for cycle number. With the use of different DNA dyes, high-end instrumentation and sophisticated analysis software, these distinctions are detected.

Keywords: Antimicrobial resistance, Genotyping, Genetic variants, HRMA, Intercalating dyes, Melting curve.