Modified High-Resolution Melting (HRM) Marker Systems Increasing Discriminability Between Homozygous Alleles

Satoshi Watanabe; Yoshiyuki Yamagata; Nobuhiro Kotoda

doi:10.1007/978-1-0716-3024-2_25

Modified High-Resolution Melting (HRM) Marker Systems Increasing Discriminability Between Homozygous Alleles

Methods Mol Biol. 2023:2638:351-363. doi: 10.1007/978-1-0716-3024-2_25.

Authors

Satoshi Watanabe¹, Yoshiyuki Yamagata², Nobuhiro Kotoda³

Affiliations

¹ Faculty of Agriculture, Saga University, Saga, Japan. nabemame@cc.saga-u.ac.jp.
² Faculty of Agriculture, Kyushu University, Fukuoka, Japan.
³ Faculty of Agriculture, Saga University, Saga, Japan.

PMID: 36781655
DOI: 10.1007/978-1-0716-3024-2_25

Abstract

Targeted single-nucleotide polymorphism (SNP) genotyping, especially for functional nucleotide polymorphism, is widely used for current breeding programs in crops. One of the cost- and time-effective approaches for genotyping is high-resolution melting (HRM) analysis for polymerase chain reaction (PCR) amplicons, including target SNP. The reliability of a genotype obtained from an HRM marker depends on the difference in T_m values between two amplicons. Increasing the reliability of HRM marker genotypes could be archived with the selection of the best nearest neighboring nucleotide substitution (NNNs) in primer sequences surrounding SNPs. This chapter provides an easy-way protocol to design primer sequences for NNNs-HRM markers with table and web service, as well as several tips to develop HRM markers that distinguish between homozygous alleles (e.g., between A/A and C/C).

Keywords: High-resolution melting (HRM) analysis; Marker-assisted selection; Molecular marker; PCR; SNPs.

MeSH terms

Alleles
Genotype
Nucleic Acid Denaturation
Nucleotides
Plant Breeding*
Polymorphism, Single Nucleotide*
Reproducibility of Results

Substances

Nucleotides