A combination of RNase H and S1 nuclease circumvents an artefact inherent to conventional S1 analysis of RNA splicing.
A combination of RNase H and S1 nuclease circumvents an artefact inherent to conventional S1 analysis of RNA splicing.
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RNase H 和 S1 核酸酶的组合避免了 RNA 剪接的常规 S1 分析固有的假象。
DOI：
10.1093/nar/15.5.1995
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发表时间：
1987
影响因子：
14.9
通讯作者：
Sollner-Webb,B
中科院分区：
文献类型：
--
作者：
Sisodia,SS;Cleveland,DW;Sollner-Webb,B
S1核酸捕捉技术常用于分析未标记RNA的转录和加工过程。然而，似乎最适合于证明特定RNA的分裂的SI方案（使用在下游外显子中3'末端标记的无内含子探针）并不像预期的那样是诊断性的。相反，含内含子的RNA和无内含子的RNA都赋予探针穿过剪接接合点的保护。为了明确地证明正确剪接的RNA，开始在一个特定的起始位点，我们提出了一个程序，其中unsplioed RNA分子首先裂解RNase B退火后的内含子DNA片段和重新获得的RNA，然后进行SI分析，使用大量过量的无内含子探针。只有分裂的、或正确起始的转录物才能保护探针不受分裂连接的影响，直到残基+1。这种RNase H/S1方法提供了一种广泛适用的技术，用它来证明各种转录本的剪接和起始，特别是来自正常和激活的转录起始位点的转染基因的转录本。
SI nucleate napping it commonly used to analyze transcription and processing of unlabellod RNAs. However, the SI protoool that appears best suited to demonstrate splioing of a particular RNA (using an intronless probe that is 3' end-labelled in the downstream exon) is not diagnostio as expeoted. Rather, both intron-oontaining RNA and intronless RNA confer protection of probe across the splice juncture. To unambiguously demonstrate correotly spliced RNAs that begin at a specific initiation site, we present a procedure in which unsplioed RNA moleoules are first cleaved by RNase B following annealing to an intronic DNA fragment and the regaining RNA is then subjected to SI analysis using an intronless probe present in vast excess. Only splioed, oorreotly initiated transcripts can protect the probe aoross the splioe junotion and up to residue +1. This RNase H/Sl Method provides a broadly applioable teohnique with which to demonstrate splicing and initiation of a variety of transcripts, especially ones fron transfeoted genes that out arise both from the normal and from activated oryptic initiation sites.