Team:TU Eindhoven/RCA/Creating


iGEM Team TU Eindhoven 2014

iGEM Team TU Eindhoven 2014

Creating Circular Rolling Circle Template

A key part in the Rolling Circle Amplification is the circular DNA template. This is the template that is constantly being read by the polymerase to produce the long repeating strands starting from the primer. Since it was not possible to order ready-made circular DNA this had to be done in the lab. For this the CircLigase II enzyme was used to create circular DNA. After this was completed any remaining linear ssDNA was removed with use of exonucleases. Results were verified on denaturing PAGE gel.

Figure 1. Picture of a denaturing PAGE (15%) gel stained with
DNA-binding stain. Denaturing conditions assure no secondary
structures are formed, so linear products travel easier through
the gel than circular products. This shows that the
circular product was indeed formed, however not all linear
template has been successfully converted or removed.


As the core concept of Rolling Circle Amplification is the continuous read-out of a piece of circular single strand DNA there is a need to make this DNA. This was done through the use of the CircLigase II enzyme. This ligase requires a strand of single strand DNA that is 5’-phosphorylated. This was ordered ready-made and PAGE purified.

Our template was added to commercially supplied reaction buffer and 2.5 MnCl along with initially 5 U/µL of CircLigase II. The ligase was allowed to react for 1 hour before heat inactivation. Afterwards Exonuclease I and III were added to digest any remaining linear DNA in solution. This was allowed to react for 45 minutes before heat inactivation. However this protocol resulted in a mixture of circular and non-circular DNA. This is shown in the figure, which is a picture of a 15% denaturing PAGE gel.

To compensate for this the remainder of the DNA was circularised according to a new protocol. The amount of CircLigase per µL was doubled to 10 U/µL. The reaction was still carried out for 1 hour. Exonuclease I and III were once more added, but now were allowed to react for 1 hour before heat inactivation. This was also loaded on a denaturing PAGE gel, however results were unclear. Any further attempts to show purity and effectiveness of this protocol have not been undertaken due to lack of time and enzyme. Also since it has been shown to provide circular DNA, the product can still be used for Rolling Circle Amplification.

For a complete protocol for this step click here for the making of the PAGE gel click here

iGEM Team TU Eindhoven 2014