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Open Access Highly Accessed Methodology article

A Strategy to Setup Codominant Microsatellite Analysis for High-Resolution-Melting-Curve-Analysis (HRM)

Eduard Mader*, Brigitte Lukas and Johannes Novak

Author Affiliations

Institute for Applied Botany and Pharmacognosy, University of Veterinary Medicine, Veterinärplatz 1, A-1210 Vienna, Austria

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BMC Genetics 2008, 9:69  doi:10.1186/1471-2156-9-69

Published: 3 November 2008

Abstract

Background

High resolution melting curve analysis (HRM) is a technique that measures exactly the decreasing fluorescence of intercalating dye in the process of dissociation of double stranded DNA. The measurement is immediately following PCR in a one-step, closed-tube method. The shape of the melting curve depends on the GC content, length and sequence of the amplicon. Hence it is a powerful, fast and cheap method to detect Single Nucleotide Polymorphisms (SNPs) and other mutations.

Results

Here we present a strategy to set up microsatellite analysis for HRM including the correct assignment of heterozygous samples by comparative analysis and artificial mixtures of samples. The approach is demonstrated on two Simple Sequence Repeat (SSR) loci of different complexity in the genus Origanum. Following this strategy all alleles of our sample sets could be classified correctly.

Conclusion

HRM can be used in microsatellite analysis and other codominant marker systems implementing a protocol of comparative melting curve assignment with artificial mixtures of samples to overcome difficulties in correctly assigning heterozygous samples. The method is faster, more sensitive and cheaper than standard protocols for microsatellite analysis.