PCR Cloning Method
Return to Cloning & Synthetic BiologyHigh-fidelity DNA polymerases are also now routinely used to amplify sequences with the PCR product containing no 3' extensions. The blunt-end fragments are joined to a plasmid vector through a typical ligation reaction or by the action of an "activated" vector that contains a covalently attached enzyme, typically Topoisomerse I, which facilitates the vector:insert joining. Some PCR cloning systems contain engineered "suicide" vectors that include a toxic gene into which the PCR product must be successfully ligated to allow propagation of the strain that takes up the recombinant molecule during transformation.
A typical drawback common to many PCR cloning methods is a dedicated vector that must be used. These vectors are typically sold by suppliers, like NEB, in a ready-to-use linearized format and can add significant expense to the total cost of cloning. Also, the use of specific vectors restricts the researcher's choice of antibiotic resistance, promoter identity, fusion partners, and other regulatory elements.
Advantages:- High efficiency, with dedicated vectors
- Amenable to high throughput
- Limited vector choices
- Higher cost
- Lack of sequence control at junction
- Multi-fragment cloning is not straight forward
- Directional cloning is difficult