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Versatile gene-specific sequence tags for Arabidopsis functional genomics: transcript profiling and reverse genetics applications.
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Microarray transcript profiling and RNA interference are two new technologies crucial for large-scale gene function studies in multicellular eukaryotes. Both rely on sequence-specific hybridization between complementary nucleic acid strands, inciting us to create a collection of gene-specific sequence tags (GSTs) representing at least 21,500 Arabidopsis genes and which are compatible with both approaches. The GSTs were carefully selected to ensure that each of them shared no significant similarity with any other region in the Arabidopsis genome. They were synthesized by PCR amplification from genomic DNA. Spotted microarrays fabricated from the GSTs show good dynamic range, specificity, and sensitivity in transcript profiling experiments. The GSTs have also been transferred to bacterial plasmid vectors via recombinational cloning protocols. These cloned GSTs constitute the ideal starting point for a variety of functional approaches, including reverse genetics. We have subcloned GSTs on a large scale into vectors designed for gene silencing in plant cells. We show that in planta expression of GST hairpin RNA results in the expected phenotypes in silenced Arabidopsis lines. These versatile GST resources provide novel and powerful tools for functional genomics.
Arabidopsis/genetics, Arabidopsis/growth &, development, Arabidopsis Proteins/genetics, DNA Primers/chemistry, DNA Primers/genetics, DNA, Plant/genetics, Databases, Genetic, Expressed Sequence Tags, Gene Expression Profiling, Gene Expression Regulation, Plant, Genome, Plant, Molecular Sequence Data, Oligonucleotide Array Sequence Analysis, Polymerase Chain Reaction, RNA Interference, RNA, Messenger/genetics, RNA, Plant/genetics
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