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Methods to Increase the Sensitivity of High Resolution Melting Single Nucleotide Polymorphism Genotyping in Malaria
Published on: November 10, 2015
A strategy to setup codominant microsatellite analysis for high-resolution-melting-curve-analysis (HRM)
Eduard Mader1, Brigitte Lukas, Johannes Novak
1Institute for Applied Botany and Pharmacognosy, University of Veterinary Medicine, Veterinärplatz 1, A-1210 Vienna, Austria. eduard.mader@vu-wien.ac.at
BMC Genetics
|November 5, 2008
Summary
High resolution melting curve analysis (HRM) offers a fast, sensitive, and cost-effective method for microsatellite analysis. This technique accurately identifies alleles, including heterozygous samples, by employing comparative analysis and artificial mixtures.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- High-resolution melting curve analysis (HRM) quantifies DNA dissociation by measuring decreasing fluorescence after PCR.
- Melting curve characteristics (GC content, amplicon length/sequence) enable detection of genetic variations like Single Nucleotide Polymorphisms (SNPs).
- HRM is a rapid, cost-effective, closed-tube method for genetic analysis.
Purpose of the Study:
- To develop and validate a strategy for microsatellite analysis using HRM.
- To accurately assign heterozygous samples in microsatellite analysis.
- To demonstrate the applicability of HRM in the genus Origanum.
Main Methods:
- Development of a microsatellite analysis protocol utilizing HRM.
- Implementation of comparative analysis and artificial sample mixtures for allele assignment.
- Application of the HRM strategy to two Simple Sequence Repeat (SSR) loci in Origanum.
Main Results:
- Successful classification of all alleles within the sample sets.
- Demonstration of accurate heterozygous sample assignment through comparative analysis.
- Validation of the HRM strategy for microsatellite analysis in Origanum.
Conclusions:
- HRM is a viable and efficient method for microsatellite analysis and other codominant marker systems.
- Comparative melting curve assignment with artificial mixtures effectively resolves heterozygous sample identification.
- HRM provides a faster, more sensitive, and cheaper alternative to standard microsatellite analysis protocols.
