The resulting double-stranded cDNAs with linkers at both ends are treated with a restriction enzyme specific for the linker generating cDNA molecules with sticky ends. Problems arise, when cDNA itself has a site for the restriction enzyme cleaving the linkers. This can be overcome using an appropriate modification enzyme (methylase) to protect any internal recognition site from digestion which methylates specific bases within the restriction-site sequence, thereby, preventing the restriction enzyme binding.

 

Figure 4-3.2.3.2: Action of methylases

Ligation of the digested ds-cDNA into a vector is the final step in the construction of a cDNA library. The vectors (e.g. plasmid or bacteriophage) should be restricted with the same restriction enzyme used for linkers. The E. coli cells are transformed with the recombinant vector, producing a library of plasmid or λ clones. These clones contain cDNA corresponding to a particular mRNA.