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Synthesis of complementary RNA on RNA templates using the DNA-dependent RNA polymerase of Escherichia coli.

作者信息

Bernard O, Cory S, Adams J M

出版信息

Biochim Biophys Acta. 1977 Oct 18;478(4):407-16. doi: 10.1016/0005-2787(77)90096-x.

DOI:10.1016/0005-2787(77)90096-x
PMID:334264
Abstract

It is shown that the DNA-dependent RNA polymerase of Escherichia coli can synthesize complementary RNA (cRNA) directly on rRNA and mRNA templates. Synthesis occurred preferentially in the presence of Mn2+ and at relatively high substrate and enzyme concentrations. No primer was required, and addition of oligo-U to a mRNA-dependent reaction gave no marked stimulation. Sedimentation analysis of cRNA made on different templates indicated that the products were mainly 2-4 S, but a fraction of the product was larger. Fingerprints of 32P-labelled cRNA made on 5 S rRNA and 18 S rRNA indicated that the complexity of the cRNAs was related to the size of the template, suggesting that a substantial portion of the templates were copied. This reaction provides a simple method for preparing cRNA of high specific activity for use in hybridisation studies, and possibly in sequence analysis. 32P-labelled cRNA made on 18 S and 28 S rRNA was a sensitive hybridisation probe for detection of the specific fragments of mouse DNA containing the rRNA genes.

摘要

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