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, the DBCO-Azido click system 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.

Summary

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.

Bibliography

Mazutis, Linas, John Gilbert, W Lloyd Ung, David A Weitz, Andrew D Griffiths and John A Heyman. Single-cell analysis and sorting using droplet-based microfluidics. Nature protocols 8.5 (2013): 870-891.