Adaptation of the differential display RT-PCR technique to isolate sugarcane genes induced by plant association with endophytic nitrogen-fixing bacteria.
Differential Display RT-PCR is a powerful technique that has been used to isolate differentially expressed genes. This technique was first described by Liang & Pardee, in 1992. Afterwards, several modifications were introduced in the original version, including a simplification described by Sokolov & Prockop, in 1994. In this work, we describe an adaptation of the Sokolov & Prockop technique, in order to isolate sugarcane genes induced by plant association with endophytic nitrogen-fixing bacteria. Several modifications were introduced: the use of oligo-dT primer in the first strand cDNA synthesis, replicates of the PCR reactions, analysis of the amplified fragments on silver stained polyacrilamide gel and confirmation of cloning the differentially amplified selected band prior to its molecular characterisation. The methodology established was successfully used to identify a large number of differentially amplified sugarcane cDNAs. So far, one of these cDNAs was already isolated and characterized.
Vargas
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Departamento de Bioquímica Médica, ICB/CCS/UFRJ, Cidade Universitária, Rio de Janeiro, Brasil.
Lopes
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Hemerly
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D018076:DNA, Complementary / Q000032:analysis*; D017343:Genes, Plant; D009586:Nitrogen Fixation; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
D018076:DNA, Complementary