4.4 More Cloning
Several variations on the techniques just presented will illustrate practical cloning solutions (see FIG. 37). In the first, a cDNA library is constructed. Instead of adding a tail of dC residues to provide a binding site for second strand primer, the investigators relied on the ability of reverse transcriptase to make hairpin turn at the 3' end of the new strand. This short hairpin now provides a primer to complete the second strand. The hairpin may be cleaved by S1 nuclease that makes cuts in single stranded regions, such as the extreme end of the hairpin would be. Another feature of this cloning is the attachment of short adapters molecules to each end of the new double stranded DNA using DNA ligase (blunt-ended ligation). These adapters contain one or more restriction sites such as the one shown for HinDIII. The plasmid also was opened with HinDIII for insertion of the DNA.
The next example (FIG. 39) illustrates the construction of a genomic library. The genomic DNA was digested with limiting amounts of HaeIII and AluI to give DNA fragments in the 10--50 Kbp range. The DNA was fractionated on agarose gels and fragments of about 20 Kbp were selected for the cloning. The fragments were methylated at sites that would protect against EcoRI action (can you explain why?), and then an adapter was added to each end with ligase. The completed molecule was digested with EcoRI to create sticky ended sites. A Charon 4A was selected and annealed to bind together the cos sites, and then the circular molecule was digested with EcoRI. The large, central fragment was isolated (containing the cos site and EcoRI sticky ends) and ligated with the DNA fragments to obtain long, linear molecules that could be packaged in vitro into new phage particles. The phage particles constitute the library, or the library may be expanded by growth in E. coli.
Figure 39. Cloning a DNA library
At this point we could prepare a cDNA library starting from pancreatic beta-cells that should contain the mRNA for proinsulin. The series of steps would be very similar to those described in an earlier section, except we would use reverse transcriptase to create the cDNA copy instead of chemically synthesizing the DNA genes for the separate A and B chains. We again would add the code for methionine so that we may split out the finished proinsulin from the beta-galactosidase fusion product using cyanogen bromide. After the proinsulin is isolated, we need to split out and discard the C peptide using protease enzymes. We also could use another approach and isolate the insulin gene from a genomic library as well.
Figure 40. Cloning the complete gene for insulin
There are special enzymes used to cleave out the C peptide that are found in pancreatic beta-cells. These special peptidases are used by the cell to specifically produce mature insulin without accumulation of unwanted side products.