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Team:TU Eindhoven/RCA/Attaching
From 2014.igem.org
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.
DNA with 12C spaced primary amine. Used to form our
clickable DNA oligo.
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.
This reaction was carried out at room temperature for two hours after which it 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%, including washing.
The resulting molecule can be used to click DNA to the cell surface which was used to initiate a rolling circle amplification reaction used to create a DNA-based hydrogel around the cell.
