Principles of cell-based cloning:
Four steps in cell-based cloning
- Construction of recombinant DNA molecules. Involves the use of endonuclease restriction enzymes, ligation, and a replicon (vector).
- Transformation in appropriate host cells.
- Selective propagation of cell clones. This step takes advantage of selectable markers.
- Isolation of recombinant DNA from cell clones followed by molecular characterization (such as restriction enzyme analysis).
Endonuclease restriction enzymes type II (RE), are a powerful tool (molecular scissors) used in restricting target DNA (whole genome or plasmid) into smaller DNA fragments. The restriction of a DNA double helix molecule may result in a blunt end or a cohesive end terminus (sticky end generating a 3’ or 5’ single strand overhang). See Table 5.1
RE are used to generate recombinant linear molecules (concatemers) or circular molecules (cyclization).
Simple cloning vectors include bacterial plasmids and bacteriophages. 
