Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 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.

PubMed Disclaimer

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.

Similar articles

See all similar articles

References

    1. Simko I (2016) High-resolution DNA melting analysis in plant research. Trends Plant Sci 21(6):528–537. https://doi.org/10.1016/j.tplants.2016.01.004 - DOI - PubMed
    1. Hjelmso MH, Hansen LH, Baelum J, Feld L, Holben WE, Jacobsen CS (2014) High-resolution melt analysis for rapid comparison of bacterial community compositions. Appl Environ Microbiol 80(12):3568–3575. https://doi.org/10.1128/AEM.03923-13 - DOI - PubMed - PMC
    1. Landolt P, Stephan R, Scherrer S (2019) Development of a new High Resolution Melting (HRM) assay for identification and differentiation of Mycobacterium tuberculosis complex samples. Sci Rep 9(1):1850. https://doi.org/10.1038/s41598-018-38243-6 - DOI - PubMed - PMC
    1. Ngui R, Lim YA, Chua KH (2012) Rapid detection and identification of human hookworm infections through high resolution melting (HRM) analysis. PLoS One 7(7):e41996. https://doi.org/10.1371/journal.pone.0041996 - DOI - PubMed - PMC
    1. Robertson T, Bibby S, O’Rourke D, Belfiore T, Agnew-Crumpton R, Noormohammadi AH (2010) Identification of Chlamydial species in crocodiles and chickens by PCR-HRM curve analysis. Vet Microbiol 145(3–4):373–379. https://doi.org/10.1016/j.vetmic.2010.04.007 - DOI - PubMed

LinkOut - more resources