RNA transcription from immobilized DNA templates.
We describe an RNA transcription protocol based on the multiple reuse of solid-phase synthetic DNA templates. The templates are assembled onto streptavidin-coated agarose beads via a single 5'-terminal biotin located on the noncoding template strand. Transcription occurs in an aqueous buffered suspension containing solid-phase DNA, dissolved enzyme (T7 RNA polymerase), and nucleoside triphosphate substrates (NTPs). A direct comparison of solution and solid-phase templates under standard transcription conditions reveals similar initial reaction rates and overall yields. Immobilized templates store stably for periods of several months and are easily recovered by mild centrifugation. We demonstrate the successive reuse of these templates throughout 15 rounds of transcription. The templates remained active, although an incremental decay in transcription was observed beyond five rounds. Template activity was partially restored by supplementing the support-bound oligonucleotide with fresh coding-strand DNA. These findings indicate that multiple reuse of template is a viable strategy for reducing the amount of DNA template required in RNA transcription.
Marble
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HA
Department of Chemical Engineering, University of Colorado, Boulder 80309-0424, USA.
Davis
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RH
D001345:Autoradiography; D001483:Base Sequence; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D012367:RNA, Viral / Q000096:biosynthesis*; D012996:Solutions; D013604:T-Phages / Q000235:genetics / Q000378:metabolism; D013698:Templates, Genetic; D014158:Transcription, Genetic
D004279:DNA, Viral; D012367:RNA, Viral; D012996:Solutions