Haplotype and Presence-Absence Variation Analysis Using High-Resolution Melting (HRM) Analysis for Genotyping Genes of Interest.

This chapter outlines a basic protocol for DNA extraction, applicable to both FTA cards and leaf pieces. The extracted DNA then undergoes a pre-amplification enrichment reaction, followed by dilution and preparation for input into a real-time amplification with High-Resolution Melt (HRM) detection. The resulting melting genotypes are produced for comparison and analysis, providing a practical approach for genotyping studies. The flexibility of the method allows specific analytical output and high throughput analysis of a given DNA sequence that possesses inherent variability between individuals. This protocol describes the crude approach of extracting DNA from samples, preparing a real-time HRM reaction, and analyzing the result.