Detection of Gene Products Expressed from Plasmids

This chapter discusses various techniques including the use of bacterial minicells, maxicells, cell free in vitro protein synthesis, and RNA detection systems including hybridization and S1 nuclease analysis. If a relatively large piece of DNA has been cloned, it is sometimes desirable to use a rapid screen to detect polypeptides or messenger RNA (mRNA) expressed from the fragment without DNA sequencing. In certain situations, immunological or enzymatic approaches can be useful aids to identifying proteins. A number of techniques have been developed in recent years that allow the detection of gene products expressed from cloned DNA. Some of the techniques to be described can also be used for the detection of chromosomally-encoded gene products. A major difficulty encountered when analysing plasmid or phage-encoded mRNA and polypeptides in whole (normal) bacterial cells is that the majority of these products are masked by those encoded by the host cells chromosome. This difficulty often remains even when the genes of interest have been amplified by cloning into multicopy vectors.