Bacteriophage hybridization

In 1961, Jacob and Monod postulated messenger RNA (mRNA) as a short-lived polynucleotide.30 32 33 An abundance of additional evidence supported the proposal. For example, RNA molecules produced after infection of E. coli by bacteriophage T4 underwent hybridization (Chapter 5) with denatured DNA of the bacteriophage. Furthermore, this virus-specific mRNA became associated with preexisting bacterial ribosomes and provided the template for synthesis of phage proteins.34 The experiment provided direct evidence for transcription of mRNA from genes of the viral DNA. 

When a bacteriophage is used as the cloning vector, the gene library is screened as an array of plaques in a bacterial lawn. A hybridization screening method is used similar to that described for plasmid screening above in this case the replica filter is called a plaque lift. ... 

Melton, D.A., P.A. Krieg, M.R. Rebagliati, T. Maniatis, K. Zinn and M.R. Green. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 12 7035-7056, 1984. 

RNase protection assays (RPA) are based on the property of RNase to digest ii RNA, but not ds RNA, and its principles and applications resemble those of SI analysis (Lynn et al., 1983 Zinn et at, 1983) (Fig. 12.3). The sequence of interest is inserted in a plasmid downstream of a bacteriophage promoter (e.g., pUC118, pT7, etc.. Table 4.4). The purified plasmid is then restricted downstream of the inserted DNA and the linearized plasmid is transcribed in the presence of a labeled rNTP precursor. The transcript should be complementary to the RNA to be studied and an excess of probe is hybridized to its target. Any RNA remaining ss is then digested by one or more RNases. The size of the RNase-resistant probe and the... 

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