Department of Veterinary Microbiology, Central Agricultural University-Imphal, Selesih, Aizawl, Mizoram, India
High Resolution Melting Analysis (HRMA) is a simple, PCR-based method for detecting DNA sequence variation by measuring changes in the melting of a DNA duplex. To follow the transition of double-stranded DNA (dsDNA) to singlestranded (ssDNA), intercalating saturating dyes such as LC Green, EvaGreen, Chromofy and SYTO 9 were employed. HRM is based on the analysis of fluorescence curves produced by DNA intercalating dye during strand dissociation events in the melting phase following real-time PCR. The melting temperature at which 50% of the DNA is in the double stranded state can be approximated by taking the derivative of the melting curve. The distinctive melting curve can be used to detect DNA sequence variants without the need for any post-PCR handling. It is a rapid, cost effective, less complicated method with high sensitivity and specificity. The strategic use of this methodology includes identification of Single Nucleotide Polymorphism (SNPs), genotyping, species identification and detection of genetic variants related to antimicrobial resistance. The simplicity, low cost and high efficiency of the technique is promising to take leading role in biological research in veterinary science in future.
Applications, High resolution melting, Principle, Veterinary
