In this exercise we will simulate TOPO-cloning using the plasmid vector pCRII-TOPO™.
ThermoFisher supplies this vector in linearized form, with covalently-attached Topoisomerase enzyme, and in the TA-vector case, with 3'-T overhangs. However, when you retrieve the vector sequence online and import it into Geneious, the vector is circular and does not have overhang modifications. There is no need to add overhangs as the TOPO cloning operation will add these automatically. The only requirements are that you have a circular vector containing a TOPO site and a compatible linear insert.
In the lab, insert fragments for TA cloning are usually amplified by PCR using Taq polymerase. Taq polymerase has a nontemplate-dependent terminal transferase activity that adds a single deoxyadenosine (A) to the 3ยด ends of PCR products. These 3'-A overhangs complement the 3'-T overhangs in a linearised TA-vector. As with the vector sequence, you do not need to add the 3'-A's to your inserts in Geneious. The TOPO cloning operation will do this automatically for you.
Exercise
First, we will use the Geneious Search for Motifs tool to find and annotate the TOPO site onto the plasmid vector.
Select the pCRII-TOPO™ sequence file in the Document table to view it. The green icon associated with this file indicates that the sequence is circular.
Copy (CTRL/command-c) the following motif CCCTTAAGGG, then go menu Annotate & Predict → Search for Motifs.... Paste the sequence in the Motif: field, set Name and Annotation Type: to TOPO. As this motif is palindromic it will be found (and annotated) on the forward and reverse strands. Uncheck the option for Forward strand so that the motif is only found and annotated onto the reverse strand of the vector. Click OK to run the Find Motif operation, then Save.
Zoom into position 340 bp and you will see the new motif which is located between two EcoRI restriction sites. Note that it is not essential to annotate the TOPO site onto your vector, Geneious will recognize the TOPO site automatically, regardless of whether it is annotated, and use it.
The Vaccinia DNA Topoisomerase I cleaves one strand of DNA after a 5'-(C/T)CCTT motif and covalently binds a resulting 3'-phosphate to its tyrosyl residue (Tyr274)[1,2]. Upon annealing to a PCR-derived insert (no 5'-phosphates) the insert-based 5'-hydroxyl attacks the exposed phosphate releasing the topoisomerase and ligating the vector and insert ends. This occurs at both ends of the linearized vector, creating a circular vector + insert.
Picture taken from Topo® TA manual, ThermoFisher Scientific
In practise TOPO-cloning is not directional and the insert will ligate in both orientations. However, in Geneious only the current orientation (defined by the orientations of documents as viewed in the sequence viewer) will be ligated into the vector. If you require both insert orientations, create a copy of the insert sequence, reverse complement it (use the R.C. button, or go menu Sequence → Reverse Complement...) and perform the TOPO cloning operation with both inserts (see section 4 Batch Cloning).
If you zoom out from the TOPO site you will see that downstream of the MCS there is a T7 Promoter pointing towards the MCS in the reverse direction. We want this promoter to be oriented in the same direction as the Human Proinsulin sequence (which is oriented in the forward direction), so that we can eventually use it to express our gene product.
Therefore, we should reverse the vector sequence before we proceed by selecting the pCRII-TOPO™ sequence and clicking on R.C. button above the sequence viewer. Choose to reverse complement entire sequence, then click OK and then Save. Zoom in and you will see that the T7 promoter and TOPO annotations are now oriented in the forward direction.
Next, select the Human Proinsulin sequence. This sequence contains an annotated CDS in the forward orientation with a short sequence before and after it. We want to include the whole sequence in our final construct, so this sequence does not need any editing in Geneious.
Next, select both the vector and proinsulin sequences, and click on Cloning → TOPO Cloning. In the options dialog select TA Cloning. You should see your vector listed in the textbox.
Click OK and the ligated product will be created, called Human proinsulin ligated into pCRII-TOPO (reversed) and saved in the Document table. Hold down the option/alt key and click on the red Insert annotation to zoom in on that region. You'll see that the proinsulin CDS has been inserted into the middle of the MCS at the the TOPO site, and if you scroll to the left you'll see the T7 promoter at the 5'-end, in the same orientation as the insert.
Use the link below to continue with Exercise 2.
Exercise 2: Cloning into ZeroBlunt Vector
Exercise 3: Directional Cloning
Exercise 4: Batch Cloning
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