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. 2023:2638:337-349.
doi: 10.1007/978-1-0716-3024-2_24.

High-Resolution Melting (HRM) Genotyping

Nayoung Kim  1 , Ji-Su Kwon  1 , Won-Hee Kang  1   2 , Seon-In Yeom  3   4
Affiliations

High-Resolution Melting (HRM) Genotyping

Nayoung Kim et al. Methods Mol Biol. 2023.

Abstract

High-resolution melting (HRM) analysis is a simple, fast, and inexpensive real-time polymerase chain reaction (PCR)-based method used to identify genetic variation between populations and detect single-nucleotide polymorphisms (SNPs) in nucleic acid sequences. HRM is a powerful technique that detects the differences between SNP allele melting temperatures by using a fluorescent dye inserted into the duplex deoxyribonucleic acid (DNA) structure. Prior to performing HRM analysis, optimizing the primer design, PCR mixture, and software settings is essential to obtain accurate and reliable results. In this chapter, we describe a detailed SNP genotyping method that includes primer design and the analysis of the shapes and positions of the melt curve of the luminescence intensity of the fluorescent dye attached to amplified DNA using software of qPCR instruments. This protocol is applicable for genotyping germplasm, genetic mapping, and marker-assisted breeding in plants.

Keywords: Genetic variation; HRM; High-resolution melting analysis; Real-time PCR; SNP genotyping; Single-nucleotide polymorphism.

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References

    1. Gundry CN, Vandersteen JG, Reed GH, Pryor RJ, Chen J, Wittwer CT (2003) Amplicon melting analysis with labeled primers: a closed-tube method for differentiating homozygotes and heterozygotes. Clin Chem 49:396–406. https://doi.org/10.1373/49.3.396 - DOI
    1. Wittwer CT, Reed GH, Gundry CN, Vandersteen JG, Pryor RJ (2003) High-resolution genotyping by amplicon melting analysis using LCGreen. Clin Chem 49:853–860. https://doi.org/10.1373/49.6.853 - DOI
    1. Tucker EJ, Huynh BL (2014) Genotyping by high-resolution melting analysis. In: Fleury D, Whitford R (eds) Crop breeding: methods and protocols, vol 1145. Humana, New York, pp 59–66. https://doi.org/10.1007/978-1-4939-0446-4_5 - DOI
    1. Park SW, An SJ, Yang HB, Kwon JK, Kang BC (2009) Optimization of high resolution melting analysis and discovery of single nucleotide polymorphism in Capsicum. Hortic Environ Biotechnol 50:31–39
    1. Kim N, Kang WH, Lee J, Yeom SI (2019) Development of clustered resistance gene analogs-based markers of resistance to Phytophthora capsici in chili pepper. Biomed Res Int 2019:1093186. https://doi.org/10.1155/2019/1093186 - DOI

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