A method for generating subtractive cDNA libraries retaining clones containing repetitive elements.
Here we describe a two-stepped photobiotin-based procedure to enrich a target (canine retinal) cDNA library for tissue specific clones without removing those containing repetitive ( SINE ) elements, despite the presence of these elements in the driver population. In a first hybridization excess SINE elements were hybridized to a driver (canine cerebellar) cDNA. In a second hybridization target cDNA was added to this reaction. The resulting cDNA library was enriched for retinal specific clones, but contained the same ratio of clones with SINE elements found in the unsubtracted library.
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Department of Clinical Veterinary Medicine, Centre for Veterinary Sciences, University of Cambridge, Madingley Road, Cambridge CB3 0ES, UK.
Sargan
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DR
D000818:Animals; D001386:Azides; D001710:Biotin / Q000031:analogs & derivatives; D002531:Cerebellum; D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary / Q000235:genetics*; D004285:Dogs; D015723:Gene Library; D009693:Nucleic Acid Hybridization; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics*; D012160:Retina
D001386:Azides; D018076:DNA, Complementary; D001710:Biotin; C045549:photobiotin