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Genetic Variant Detection in the CALR gene using High Resolution Melting Analysis

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

Anthony Torres¹, Robert Givens², Reginald Gaudino^1,2

¹Cannabis Research Institute, Discovery Partners Institute, University of Illinois System, Chicago, IL, USA.

Methods in Molecular Biology (Clifton, N.J.)

|June 28, 2025

Summary

This study presents a DNA extraction and genotyping protocol using High-Resolution Melt (HRM) analysis for efficient individual DNA sequence comparison. The method enables high-throughput genotyping studies with practical DNA analysis.

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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Area of Science:

Molecular Biology
Genetics

Background:

DNA extraction is a foundational step in molecular biology.
High-Resolution Melt (HRM) analysis is a powerful post-PCR technique for DNA melt curve analysis.

Purpose of the Study:

To outline a basic protocol for DNA extraction and subsequent genotyping using HRM analysis.
To provide a practical and flexible method for high-throughput genotyping studies.

Main Methods:

DNA extraction from FTA cards and leaf tissues.
Pre-amplification enrichment, dilution, and real-time amplification.
High-Resolution Melt (HRM) detection for genotype analysis.

Main Results:

Successful DNA extraction from various sample types.
Generation of melting genotypes for comparison and analysis.
Demonstration of a flexible approach for DNA sequence variability analysis.

Conclusions:

The described protocol offers a practical approach for DNA extraction and genotyping.
The method supports high-throughput analysis of DNA sequences with inter-individual variability.
This protocol is applicable to various genotyping studies.