Melton D A, Krieg P A, Rebagliati M R, Maniatis T, Zinn K, Green M R
Nucleic Acids Res. 1984 Sep 25;12(18):7035-56. doi: 10.1093/nar/12.18.7035.
A simple and efficient method for synthesizing pure single stranded RNAs of virtually any structure is described. This in vitro transcription system is based on the unusually specific RNA synthesis by bacteriophage SP6 RNA polymerase which initiates transcription exclusively at an SP6 promoter. We have constructed convenient cloning vectors that contain an SP6 promoter immediately upstream from a polylinker sequence. Using these SP6 vectors, optimal conditions have been established for in vitro RNA synthesis. The advantages and uses of SP6 derived RNAs as probes for nucleic acid blot and solution hybridizations are demonstrated. We show that single stranded RNA probes of a high specific activity are easy to prepare and can significantly increase the sensitivity of nucleic acid hybridization methods. Furthermore, the SP6 transcription system can be used to prepare RNA substrates for studies on RNA processing (1,5,9) and translation (see accompanying paper).
本文描述了一种简单高效的方法，可用于合成几乎任何结构的纯单链RNA。该体外转录系统基于噬菌体SP6 RNA聚合酶异常特异性的RNA合成，它仅在SP6启动子处起始转录。我们构建了方便的克隆载体，其在多克隆位点序列的紧邻上游含有一个SP6启动子。利用这些SP6载体，已确定了体外RNA合成的最佳条件。证明了SP6衍生的RNA作为核酸印迹和溶液杂交探针的优点及用途。我们表明，高比活性的单链RNA探针易于制备，并且可显著提高核酸杂交方法的灵敏度。此外，SP6转录系统可用于制备用于RNA加工（1,5,9）和翻译研究的RNA底物（见随附论文）。
