Joining of long double-stranded RNA molecules through controlled overhangs.
We describe two methods for creating long (>1 kb) dsRNA molecules with specific, user-controlled overhangs for efficient hybridization and ligation. The two methods create double-stranded RNA (dsRNA) molecules with 5' overhangs or with 3' overhangs using T7 RNA polymerase (T7 RNAP) in transcription reactions of carefully designed PCR products. Primers utilized in the PCR reactions provide the template for the desired dsRNA overhangs. These methods provide complete control of the length and the sequence of the overhangs. This supplies a tool which is particularly lacking in dsRNA biochemistry given the absence of restriction endonucleases active on these substrates.
Kavli Institute of Nanoscience, Faculty of Applied Sciences, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The Netherlands. Nynke.Dekker@mb.tn.tudelft.nl
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Veenhuizen
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Bruinink
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D004247:DNA / Q000737:chemistry; D017931:DNA Primers; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction; D012330:RNA, Double-Stranded / Q000737:chemistry* / Q000378:metabolism; D013698:Templates, Genetic; D014158:Transcription, Genetic; D014764:Viral Proteins
D017931:DNA Primers; D012330:RNA, Double-Stranded; D014764:Viral Proteins; D004247:DNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases