Avaliação de critérios de compatibilidade entre pares de primers para otimização de sistemas multiplex de genotipagem

• Ano de defesa: 2010
• Autor(a) principal: BARBOSA, Ana Clara de Oliveira Ferraz
• Orientador(a): COELHO, Alexandre Siqueira Guedes
• Tipo de documento: Dissertação
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de Goiás
• Programa de Pós-Graduação: Mestrado em Biologia
• Departamento: Ciências Biolóicas
• País: BR
• Palavras-chave em Português: marcadores moleculares; microssatélites; genotipagem; multiplex; multiplexer; 1.Marcadores moleculares 2.microssatélites 3.Genotipagem 4.Multiplex 5.Multiplexer
• Palavras-chave em Inglês: molecular markers; microsatellites; genotyping; multiplex; multiplexer
• Área do conhecimento CNPq: CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::BIOLOGIA MOLECULAR
• Link de acesso: http://repositorio.bc.ufg.br/tede/handle/tde/1291

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.