在锤头型核酶下游增加一段核酶作用的靶序列,使之成为自切割的核酶.将自切割核酶基因合成,扩增并克隆在质粒pBluescript SK上, 经连续4次克隆获得含10个拷贝的自切割核酶基因的载体. 5%聚丙烯酰胺变性凝胶电泳结果显示:体外转录过程中多体酶发生了自切割,核酶转录物切割成从70 nt到706 nt的RNA等梯度带,因此,可以作为RNA分子质量标记之用.
A self-cleaving ribozyme was comprised of a hammerhead ribozyme and its target sequence located the downstream of the hammerhead ribozyme. The self-cleaving ribozyme gene was synthesized, amplified and cloned into the Bluescript SK plasmid. The construct harboring 10 copies of the self-cleaving ribozyme gene was obtained through successively cloning for four times. The result of polyacrylamid denatured gel electrophoresis showed: the multimeric ribozymes caused self-cleaving during the transcription reaction in vitro and formed RNA step ladders from 70 nt to 706 nt, which indicates that the self-cleavage ribozyme transcripts can be used as RNA molecular mass markers.
邓文生,杨希才,康良仪,张奉学,符林春.快速制备RNA小分子质量标记的新方法[J].生物化学与生物物理进展,2000,27(3):309-311
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