Principles of Biochemistry, Volume 3 - A. Lehninger 1985

Molecular Mechanisms of Genetic Information Transfer
More on Genes: Repair, Mutation, Recombination, and Cloning
Construction of a Gene-Bearing Vector

The cDNA obtained by the method described above can now be inserted into a plasmid or a viral vector. To incorporate the cDNA into a plasmid, it must be provided with appropriate tails or sticky ends (Fig. 30-21). This is best achieved by attaching a series of repeating deoxynucleotide residues of a single type, such as A residues, to the opposite 3' ends of the two cDNA strands. These are added using terminal transferase, which, in the presence of dATP, extends both ends of the double-stranded cDNA with a poly(A) tail consisting of 50–100 residues. The cDNA is then ready for insertion into a plasmid vector (Fig. 30-21).

First, the plasmid is linearized by cleaving it at a single site with a restriction endonuclease to generate blunt ends at the Cleavage site (Section 27.24). Next, the 3' ends of the linearized plasmid are fitted with tails complementary to those added to the cDNA molecules. If the cDNA molecules were extended with poly(A) tails, the plasmid should be fitted with poly(T) 3' tails. The tailed linear plasmid and the cDNA are then simply mixed and allowed to form Base Pairs via their complementary sequences. Among the products of this Hybridization will be an enlarged circular plasmid containing the new Gene, which is held to the plasmid solely by the complementary interaction of the sticky ends. These ends can now be covalently joined using DNA ligase (Section 28.12), yielding a covalently closed circular plasmid carrying the new gene (Fig. 30-22).