Designing the Forward PCR Primer for Directional TOPO® Cloning Successful directional TOPO® Cloning depends on the design of the forward PCR primer and, to a lesser extent, on the design of the reverse PCR primer. To clone directionally, the forward PCR primer must contain a simple Kozak sequence (CACCATG) at the 5´ end of the primer.
The four nucleotides, CACC, base pair with the overhang sequence, GTGG. The bold ATG is the initiation codon of your protein of interest.
Designing the Reverse PCR Primer
To ensure that your ORF clones directionally with high efficiency, the reverse PCR primer must not be complementary to the overhang sequence GTGG at the 5´ end. A one base pair mismatch will reduce the directional cloning efficiency to 75%, and may result in your ORF being cloned in the opposite orientation. We have not observed any evidence of PCR products cloning in the opposite orientation because of a two base pair mismatch, but this has not been tested directly. Other options to consider are listed in Table 1.
In addition to the major considerations above, you may have other options to consider depending on the directional cloning vector you are using. Please refer to the respective manuals for detailed information.
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