Team:TU Eindhoven/RCA/Creating

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                   <h2>Attaching DNA to DBCO-PEG<sub>4</sub></h2>
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                   <h2>Creating Circular Rolling Circle Template</h2>
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                   <p>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-PEG<sub>4</sub> linker molecule. This is the building block that is used to click DNA to the cells and start a Rolling Circle Amplification reaction.</p>
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                   <p>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.</p>
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<h3>Summary</h3>
<h3>Summary</h3>
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<p>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-PEG<sub>4</sub> molecule to the oligo required for Rolling Circle Amplification.
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<p>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. We chose to do so ourselves with 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.
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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.</p>
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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.
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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.
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<figcaption style="font-size:18px;color:#CCCCCC;">Figure 2.</figcaption>
<figcaption style="font-size:18px;color:#CCCCCC;">Figure 2.</figcaption>
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<p>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.
 
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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.
 
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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.
 
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<h4>Bibliography</h4>
 
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<p>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.</p>
 
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Revision as of 09:51, 15 October 2014

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.

Summary

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. We chose to do so ourselves with 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.

Figure 2.

iGEM Team TU Eindhoven 2014

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