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nuclease protection assay
Nuclease protection assay is a laboratory technique used in biochemistry and genetics to identify individual RNA molecules in a heterogeneous RNA sample extracted from cells. The technique can identify one or more RNA molecules of known sequence even at low total concentration. The extracted RNA is first mixed with antisense RNA or DNA probes that are complementary to the sequence or sequences of interest and the complementary strands are hybridized to form double-stranded RNA (or a DNA-RNA hybrid). The mixture is then exposed to ribonucleases that specifically cleave only ''single''-stranded RNA but have no activity against double-stranded RNA. When the reaction runs to completion, susceptible RNA regions are degraded to very short oligomers or to individual nucleotides; the surviving RNA fragments are those that were complementary to the added antisense strand and thus contained the sequence of interest.
== Probe ==
The probes are prepared by cloning part of the gene of interest in a vector under the control of any of the following promoters, SP6, T7 or T3. These promoters are recognized by DNA dependent RNA polymerases originally characterized from bacteriophages. The probes produced are radioactive as they are prepared by in vitro transcription using radioactive UTPs. Uncomplemented DNA or RNA is cleaved off by nucleases. When the probe is a DNA molecule, S1 nuclease is used; when the probe is RNA, any single-strand-specific ribonuclease can be used. Thus the surviving probe-mRNA complement is simply detected by autoradiography.
抄文引用元・出典: フリー百科事典『 ウィキペディア（Wikipedia）』
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