Traditional restriction enzyme cloning methods are time-consuming and tedious, and with their relatively low efficiencies, multiple colonies must be screened to find the insert of interest. By removing laborious and unreliable steps from standard cloning procedures, TOPO® PCR cloning enables benchtop cloning reactions in 5 min and obtains up to 95% recombinants (Table 1). The use of TOPO® cloning vectors has been reported in the literature more than 10,000 times since the technology was launched.Table 1–Efficient subcloning with TOPO® cloning kits. Various PCR products were cloned into the pCR®2.1-TOPO® vector, transformed into One Shot®TOP10 Chemically Competent E. coli, and plated onto X-gal/amp plates. The percent recombinants represent the numbers of white colonies/the number of total colonies.
95% Cloning Efficiency in Three Steps
Invitrogen's TOPO® cloning technology harnesses the activity of DNA topoisomerase I, which in nature functions both as a restriction enzyme and as a ligase. It cleaves and rejoins DNA during Vaccinia virus replication. Topoisomerase I specifically recognizes the pentameric sequence 5′-(C/T)CCTT-3′ and forms a covalent bond with the phosphate group of the 3′ thymidine. It then cleaves one DNA strand, enabling the DNA to unwind. The enzyme then religates the ends of the cleaved strand and releases itself from the DNA. To harness the re-ligating activity of topoisomerase I, TOPO® vectors are provided linearized with topoisomerase I covalently bound to each 3′ phosphate. This enables the vectors to efficiently ligate linear DNA fragments with compatible ends. The ligation is completed in only 5 min at room temperature.
TOPO® PCR cloning only requires three steps: combine PCR product and a TOPO® cloning vector, incubate 5 min at room temperature, and transform E. coli (Figure 1). Cloning can be done 2 days faster by using TOPO® cloning kits with Mach1™-T1R competent cells compared to traditional restriction enzyme cloning (Table 2).Figure 1.
Versatility of TOPO® PCR cloning
TOPO® cloning solutions are available for use with general subcloning, sequencing, or in vitro transcription; expression in E. coli or mammalian cells; or using the Gateway® system. The power of the technology enables the pcDNA™3.3-TOPO®–adapted mammalian expression vectors to yield tens of milligrams of recombinant protein expression in suspension-adapted mammalian cells.
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