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KMID : 0613820050150050755
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Journal of Life Science
2005 Volume.15 No. 5 p.755 ~ p.762
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In vitro Synthesis of Ribonucleic Acids by T7 RNA Polymerase That was Fast Purified with a Modified Procedure
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Kim Ki-Sun
Choi Woo-Hyung
Gong Soo-Jung
Jeon Seong-Soo
Kim Jae-Hyun
Oh Sang-Taek
Kim Dong-Eun
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Abstract
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Biochemical amounts of RNA molecules can be synthesized in vitro, which is functionally equivalent or similar to those transcripts normally existing at extremely low levels in vivo. In this study we described a method for efficient preparation of pure T7 RNA polymerase from Escherichia coli strain BL21/pAR1219. The procedure, which used ammonium sulfate fractionation and preparative column chromatography on sephadex SP, was shown to be simple, rapid, and cost effective in comparison with other methods reported previously. Using the purified T7 RNA polymerase we were able to synthesize very long RNA transcript of 1.54 kb length, which is not feasible by conventional chemical synthesis. RNA molecule that was also synthesized by the purified T7 RNA polymerase, such as hammerhead ribozyme, retained its biochemical activity by cleaving the target RNA successfully in vitro. Thus, the procedure shown in this study can be useful to synthesize any length of RNA molecules in vitro in a simple and cost effective way for a variety of purposes.
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