Yang H, Melera P W
Department of Biological Chemistry, University of Maryland School of Medicine, Baltimore 21201.
Biotechniques. 1992 Dec;13(6):922-7.
We have developed a modified RNase protection assay in which the antisense RNA probe is prepared from a PCR-amplified DNA template rather than from a linearized plasmid DNA template. In this assay, an RNA polymerase promoter sequence is attached to the 5' end of the antisense PCR primer. Using this modified antisense primer in conjunction with the paired sense primer, PCR amplification generates a linear DNA template that includes an RNA polymerase promoter sequence. Transcription in vitro initiated by the incorporated promoter in the presence of RNA polymerase and ribonucleotide triphosphates produces a radiolabeled run-off antisense RNA transcript, which can then be used as probe for RNase protection analysis. Probes generated by this method obviate the need to subclone DNA sequences into transcription vectors for synthesis of antisense transcripts. Due to the simplicity of its design and the lack of need for subcloning, this strategy offers greater flexibility than conventional methods for the production of single-stranded RNA probes, and thus facilitates the implementation of the ribonuclease protection assay.
我们开发了一种改良的核糖核酸酶保护分析方法，其中反义RNA探针是由PCR扩增的DNA模板制备而成，而非来自线性化的质粒DNA模板。在该分析方法中，RNA聚合酶启动子序列连接到反义PCR引物的5'端。使用这种改良的反义引物与配对的正义引物一起，PCR扩增产生一个线性DNA模板，该模板包含一个RNA聚合酶启动子序列。在RNA聚合酶和核糖核苷酸三磷酸存在的情况下，由掺入的启动子引发的体外转录产生放射性标记的延伸反义RNA转录本，然后可将其用作核糖核酸酶保护分析的探针。通过这种方法产生的探针无需将DNA序列亚克隆到转录载体中以合成反义转录本。由于其设计简单且无需亚克隆，该策略比传统的单链RNA探针生产方法具有更大的灵活性，从而便于实施核糖核酸酶保护分析。
