chapter  4
24 Pages

Cloning

When mRNAs are converted into DNA by the enzyme reverse transcriptase (see Chapter 5), the cDNA can be incorporated or inserted into specific sites within an infectious vector, usually a virus or bacteriophage, or even into an extrachromosomal genetic element (plasmid) found among various strains of bacteria. The insertion sites are selected by identifying DNA sequences that can be cut by the actions of restriction endonucleases (see Chapter 5), enzymes from purified bacterial sources that cleave DNA sequences at specific palindromically repeated sequence sites. By using plasmids of known DNA sequence, tailored to include restriction cleavage sites that allow for DNA insertion, the same enzyme can later be used to, again, cleave out the insert. Restriction sites for insertion are typically chosen within plasmid genes that code for some discernible functional property (such as antibiotic resistance). Thus, when insertion has been successful, interruption of coding and expression leads to the loss of functional property and permits the identification of plasmids with effective inserts. In general, each plasmid can only incorporate one cDNA insert, and with a great excess of host bacteria, each insert-bearing plasmid will infect only a single host, usually

E. coli.