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. 2025:2943:131-141.
doi: 10.1007/978-1-0716-4642-7_11.

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

Anthony Torres  1 , Robert Givens  2 , Reginald Gaudino  1   2
Affiliations

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

Anthony Torres et al. Methods Mol Biol. 2025.

Abstract

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.

Keywords: Allele; Amplicon; DNA; DNA extraction; FTA cards; Genotyping; HRM; Melting difference; Melting temp; Pre-amplification; Primers; Probe; Real-time HRM; Structural and allelic variations; Tm; qPCR.

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Conflict of interest statement

Conflict of Interest. The authors have no conflicts of interest to declare that are relevant to the content of this chapter.

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