Team:TU Eindhoven/RCA/Attaching

Attaching DNA to DBCO-PEG₄

For the Rolling Circle Amplification to be used on the surface of cells a DNA-primer has to be covalently linked to the cells. This primer can then be used to start the amplification of the DNA-strand to form the long strands needed for Multiple Chain Amplification. In order to attach DNA to the cell surface, Click Coli developed was used. The steps described focus on attaching a short DNA oligo to a DBCO-PEG₄ linker molecule. This is the building block that is used to click DNA to the cells and start a Rolling Circle Amplification reaction.

Overview

In order to attach DNA-primers to a cell the click-system developed by Team Eindhoven 2014 can be used. For this to work the primer needs to be functionalized with a DBCO-group. This was done by attaching a DBCO-PEG₄ molecule to the oligo required for Rolling Circle Amplification.

To attach the molecules which normally don’t react together, DBCO-PEG₄-NHS ester was ordered as well as a 5’-amine with a twelve carbon spacer functionalized primer. These two molecules where reacted for two hours at room temperature to form the product.

After the reaction was completed the product was analysed on a 15% PAGE gel. On the gel it clearly showed that the product after two hours was heavier, thus indicating that DNA had reacted with DBCO-PEG₄-NHS ester.

The final step was to purify the product and remove any unreacted DBCO-PEG₄-NHS ester. This was done through ethanol precipitation of DNA. Next, product concentration in 100 µL MilliQ water was determined. The final concentration was used to calculate the yield of this reaction step, which was around 65% after washing.

The resulting molecule can be used to click DNA to the cell surface which was used to initiate a Rolling Circle Amplification reaction on the cell.

For a complete protocol for this step click here the protocol for the PAGE gel can be found here.