Avaliação de critérios de compatibilidade entre pares de primers para otimização de sistemas multiplex de genotipagem
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2010-01-12
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Universidade Federal de Goiás
Resumo
The progress of Molecular Biology and Genetics provided the appearance of
several molecular markers that detect the genetic polymorphism directly at DNA.
Among these markers are the microsatellites (SSR), which are distinguished by their
high degree of polymorphism. The use of these markers for individual genotyping has
evolved into multiplex systems, which allow many SSR fragments to be detected and
analyzed simultaneously. Currently there are several articles in literature discussing
the criteria to be used in the primer design for use in PCR, as well as various
softwares are available for this end. However, there are few studies and tools for the
analysis of compatibility between pairs of primers for use in multiplex systems,
where multiple fragments are simultaneously amplified using PCR. This paper
evaluated different criteria for compatibility between pairs of primers. A set of 74
combinations of pairs of primers, involving the amplification of 94 SSR loci were
evaluated in duplex systems. The same combinations were evaluated according to
different criteria, including the degree of complementarity between primers, the
magnitude of differences of denaturation temperatures (Tm) and the tendency to
annealing between pairs of primers based on the Gibbs free energy resulting from the
association between them. The comparison between the different criteria allowed the
identification of a set of criteria with positive predictive value equal to 94%. These
criteria were implemented for use in a software called Multiplexer, which from the analysis in sequence of pairs of primers, suggests compatible combinations for use in
multiplex genotyping systems. Using this tool can significantly reduce the costs
related to laboratory activities for genotyping using PCR.
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Citação
BARBOSA, Ana Clara de Oliveira Ferraz. Evaluation of compatibility criteria among primers pairs
for optimizing multiplex genotyping systems. 2010. 77 f. Dissertação (Mestrado em Ciências Biolóicas) - Universidade Federal de Goiás, Goiânia, 2010.