A Strategy to Setup Codominant Microsatellite Analysis for High-Resolution-Melting-Curve-Analysis HRMReportar como inadecuado




A Strategy to Setup Codominant Microsatellite Analysis for High-Resolution-Melting-Curve-Analysis HRM - Descarga este documento en PDF. Documentación en PDF para descargar gratis. Disponible también para leer online.

BMC Genetics

, 9:69

First Online: 03 November 2008Received: 03 July 2008Accepted: 03 November 2008

Abstract

BackgroundHigh 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.

ResultsHere 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.

ConclusionHRM 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.

Electronic supplementary materialThe online version of this article doi:10.1186-1471-2156-9-69 contains supplementary material, which is available to authorized users.

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Autor: Eduard Mader - Brigitte Lukas - Johannes Novak

Fuente: https://link.springer.com/article/10.1186/1471-2156-9-69







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