We employ the same thermostable polymerase for both PCRs, so inexperienced users can clone efficiently without mastering the idiosyncrasies of multiple restriction enzymes, polymerases, glycosylases, recombinases, and ligases.Īn outline of the overlap extension PCR cloning This relaxed double-stranded plasmid is then transformed into competent Escherichia coli cells, which seal the nicks with DNA repair enzymes ( Figure 1C). Therefore, the final product of the reaction is a double-stranded fusion plasmid with two nicks (one on each strand). F-530 New England BioLabs, Ipswich, MA, USA), crucial for performance of the technique, does not possess strand displacement activity. After denaturation and annealing, the insert strands hybridize to the vector and extend to form new double-stranded plasmid. These extensions subsequently allow the strands of the PCR product ( Figure 1A) to act as a pair of oversized primers on the vector fragment ( Figure 1B). The first of two PCRs ( Figure 1A) creates a linear insert with plasmid sequences at both ends (see Supplementary Materials for methods and instructions for primer design). 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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