Plant or fungal sequences? An alternative optimized PCR protocol to avoid ITS (nrDNA) misamplification

Brazilian Archives of Biology and Technology

Vitor Fernandes Oliveira De Miranda²  Vanderlei Geraldo Martins¹  Antonio Furlan¹  Maurício Bacci Jr.¹ 

[1] ,Universidade de Mogi das Cruzes Laboratório de Sistemática Vegetal e Herbarium Mogiense Mogi das Cruzes SP ,Brasil

关键词: polymerase;    DNA;    Drosera;    fungi;    phylogeny;   

DOI  :  10.1590/S1516-89132010000100018

来源: SciELO

【 摘 要 】

The nuclear ribosomal DNA internal transcribed spacers (ITS1 and ITS2) from leaves of Drosera (Droseraceae) were amplified using "universal" primers. The analysis of the products demonstrated most samples were a molecular mixture as a result of unsuccessful and non-specific amplifications. Among the obtained sequences, two were from Basidiomycota fungi. Homologous sequences of Basidiomycota were obtained from GenBank database and added to a data set with sequences from Drosera leaves. Parsimony analysis demonstrated that one sequence was amplified from an Ustilaginomycetes fungus, and another from a Heterobasidiomycetes. Possibly these fungi were associated to leaves of Drosera, and not because of samples contamination. In order to provide optimization and a better specificity of PCR (polymerase chain reaction), a very successful method was demonstrated using dimethyl sulfoxide (DMSO) and bovine serum albumin (BSA) in reactions.

【 授权许可】

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