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GrainGenes Reference Report: PNA-99-2146

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Reference	PNA-99-2146
Title	T-DNA integration into the barley genome from single and double cassette vectors
Journal	Proceedings of the National Academy of Sciences, USA
Year	2002
Volume	99
Pages	2146-2151
Author	Stahl R [ Show all 6 ]
Abstract	Summary: Patterns and sites of T-DNA integrations into the barley genome from single and double cassette vectors are of interest for the identification of cultivars with value added properties as well as for the production of selection marker-free transgenic lines that can be retransformed. T-DNA/Plant DNA junctions were obtained by capturing a single-stranded DNA with a biotinylated primer annealing to the vector adjacent to the border and an adaptor ligated to a restriction site overhang in the flanking barley DNA. The captured junction was converted into a double strand and sequenced. Fifty left and right border junctions from plants transgenic for one of five human genes were analyzed. Primers of 15-30 nucleotides designed from the genomic DNA at the insertion site can PCR amplify fragments that identify unequivocally any transformant. Adjacent transgene insertions with single cassette vectors were always in tandem direct repeat configuration. With regard to T-DNA integration the patterns were comparable to the variations found in dicotyledonous plants. Twelve of the 46 integrations characterized by BLAST searches were within different regions of the BARE-1 retrotransposon element occurring with a frequency of 2 x 10(5) copies in the barley genome. The use of border junctions to identify number of copies and loci of integrates in transformants is discussed
External Databases	Pubmed: 11854511
Keyword	[ Hide all but 1 of 51 ] agrobacterium tumefaciens aleurone cells alpha-amylase bare-1 bare-1 retrotransposon barley barley genome blast copies cultivar dicotyledonous plants direct repeat dna element fragments frequencies gene genome genomic dna hordeum vulgare l human insertion integration loci marker-free nucleotide pattern pcr plant dna primer production properties region repeat restriction retrotransposon search selection single-stranded-dna site t-dna t-dna integration tandem target tobacco chromosome transformation transgene transgenic vector vectors washington

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