It is, however, relatively straightforward, efficient, and reliable. Overlap extension PCR cloning, described here, is not the first form of PCR-mediated cloning ( 8–10). Recombinases are generally sold as proprietary components of cloning kits, so few consumers optimize the in vitro recombination reactions. TA cloning and LIC require end modifications that cannot be monitored by gel electrophoresis.
The methods that are easiest to monitor and optimize ultimately prove the most reliable. The practical utility of any cloning method is predicated upon its reliability, rather than its convenience, price, or efficiency under optimum conditions. Numerous alternative approaches to PCR cloning ( 1) have been developed, including TA cloning ( 2), ligation independent cloning (LIC) ( 3–4), recombinase-dependent cloning ( 5–7), and PCR-mediated cloning ( 8–10).
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