Determination of Globin mRNA Sequences and Their Insertion into Bacterial Plasmids

Publisher Summary This chapter reviews that cDNA serves as the template for synthesis of a variety of products used in various phases. The synthesis products include 32 P-labeled RNA for sequence analysis of fragments resulting from a cleavage at G residues, 32 P-labeled dC- or dT-substituted RNAs for specific cleavage at U or at C residues, and duplex gene copies for insertion into bacterial plasmids. It reviews that molecular cloning approach is applied to a wide variety of eukaryotic mRNAs. One of the advantages of the technique that is developed for the insertion of eukaryotic mRNA copies into bacterial plasmids is that a rigorous purification is not necessary; the act of cloning such material definitively eliminates all contaminating eukaryotic nucleic acid sequences. This should be especially useful to the researcher who, for instance, has a mixture of 10 different mRNA species that are difficult to separate. To obtain the corresponding sequences in pure form, it suffices to make cDNA copies of the mixture of mRNAs, convert this mixture of cDNAs to duplex gene copies, insert the DNA into bacterial plasmids and introduce the mixture of plasmids thus obtained into bacteria. Since each bacterial transformant can receive only one of the eukaryotic gene inserts, analysis of a few dozen bacterial clones should suffice to provide one with a clone for each of the interesting sequences. With such clones, it is possible to produce milligram quantities of these sequences in duplex form, sufficient for the most rapid sequencing techniques.