By Richard J. Reece
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Additional info for Analysis of Genes and Genomes
This places a limit on the times such a filter may be used. (NB. This problem becomes particularly severe if the entire length of a DNA fragment hybridizes to a complementary RNA (Haas et al, 1973). ) Single-stranded DNA and RNA can be covalently coupled to cellulose containing matrices in such a way as to be available for hybridization (for a review see Goldberg et al, 1979). Finely divided cellulose was first used as a supporting matrix (Noyes and Stark, 1975) but it was subsequently shown that paper could be chemically activated in the same way (Alwine et al, 1977), and this provides a much more convenient support (Stark and Williams, 1979).
The various positive selection procedures which have normally been used all involve a series of common steps. Firstly, the plasmid DNA is hybridized to its complementary mRNA, then unhybridized mRNA is removed in some way, and finally the mRNA is recovered from its hybrid with plasmid DNA and translated in an in vitro translation system. The hybridizaiton step may be performed in one of two different ways. With the plasmid DNA immobilized on an inert support. This is simply a modification of the classical analytical technique of Gillespie and Spiegelman (1965).
50 J. G. Williams If a particular eukaryotic protein possesses some biological or pharmacological properties, which allow detection in an extract from bacterial cells, then it might prove possible to identify a clone producing the protein. Provided the test were sensitive enough, it might even be possible to detect such a clone when it was present as part of a pool of producing and non-producing clones, hence simplifying its isolation. A cDNA clone producing a very biologically active protein, such as interferon, might potentially be isolated using such an approach, since Nagata et al.
Analysis of Genes and Genomes by Richard J. Reece