A strategy to setup codominant microsatellite analysis for high-resolution-melting-curve-analysis (HRM).
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.


CONCLUSIONS
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.
Mader
Eduard
E
Institute for Applied Botany and Pharmacognosy, University of Veterinary Medicine, Veterinärplatz 1, A-1210 Vienna, Austria. eduard.mader@vu-wien.ac.at
Lukas
Brigitte
B
Novak
Johannes
J
D000483:Alleles; D004247:DNA / Q000235:genetics; D018895:Microsatellite Repeats / Q000235:genetics*; D018598:Minisatellite Repeats; D009691:Nucleic Acid Denaturation; D031341:Origanum / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D020641:Polymorphism, Single Nucleotide; D017422:Sequence Analysis, DNA / Q000379:methods*; D013696:Temperature
D004247:DNA