The differential display technique using short primers is not suited for the routine isolation of differentially expressed sequences in sugarcane

Groenewald S. ; Botha F.C. (1999)

Article

A mRNA differential display technique using decamer primers was evaluated for the identification and isolation of differentially expressed gene sequences in the sugarcane culm. RNA was isolated from leafroll, leaf, mature culm and young culm tissues and reverse transcribed to cDNA. A series of 120 random decamer primers were used to amplify 1 767 fragments from the cDNA, resulting in an average of 15 fragments per primer. Thirty-five (2%) of these fragments were possible culm-specific sequences, and four of these were identified as putative culm-preferential rather than culm-specific fragments. None of these four fragments had significant sequence homology to sequences in the international databases. One of the fragments, SA11, was analysed using longer sequence-specific primers. Amplification products from cDNA and genomic DNA templates with these primers were of identical size. Results of a series of control reactions showed that the synthesis of the SA11 fragment from RNA was reverse transcription-dependent, and was not a product of genomic DNA contamination. Collectively the data indicate that this technique is not suitable for routine application in plants, especially those with complex genomes.

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