CLONING OF RNA MOLECULES INVITRO
CLONING OF RNA MOLECULES INVITRO
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DOI：
10.1093/nar/21.10.2349
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发表时间：
1993-05-25
影响因子：
14.9
通讯作者：
CHETVERIN, AB
中科院分区：
文献类型：
--
作者：
CHETVERINA, HV;CHETVERIN, AB
描述了一种在固定化介质中进行RNA扩增的方法。该培养基含有一套完整的核苷酸底物和纯化的Qbeta复制酶，一种能够在等温条件下以指数方式扩增RNA的酶。固定化培养基中的RNA扩增导致形成单独的“菌落”，每个菌落包含单个RNA分子的后代（克隆）。通过用溴化乙锭染色、利用放射性底物和与序列特异性标记探针杂交来观察菌落。RNA集落的数量和身份对应于接种的RNA。当接种不同RNA种类的混合物时，在不同的菌落中发现这些种类。该技术的可能实现包括重组RNA的搜索、非常灵敏的核酸诊断和体外基因克隆。
A method for RNA amplification in an immobilized medium is described. The medium contains a complete set of nucleotide substrates and purified Qbeta replicase, an enzyme capable of exponentially amplifying RNAs under isothermal conditions. RNA amplification in the immobilized medium results in the formation of separate 'colonies', each comprising the progeny of a single RNA molecule (a clone). The colonies were visualized by staining with ethidium bromide, by utilizing radioactive substrates, and by hybridization with sequence-specific labeled probes. The number and identity of the RNA colonies corresponded to that of the RNAs seeded. When a mixture of different RNA species was seeded, these species were found in different colonies. Possible implementations of this technique include a search for recombinant RNAs, very sensitive nucleic acid diagnostics, and gene cloning in vitro.