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Team:TU Eindhoven/RCA/RCA on Cells
From 2014.igem.org
Rolling Circle Amplification on Cells
As mentioned in the introduction, with Rolling Circle Amplification a long piece of single strand DNA is generated by repeating the sequence of a circular strand multiple times. Since all necessary parts are now available to test this out on clickable cells that is what was done next. Cells expressing COMPx were reacted with the DBCO coupled DNA after which the circular template was added. Rolling Circle Amplification was then carried out with the Phi29 polymerase. Finally, to verify longer strands were indeed formed for longer reaction times a fluorescent probe was added to the samples. Relative fluorescence was then measured using FACS.
different times of incubating for Rolling Circle Amplification.
The median is preferred over the average because of the
logarithmic scale used in FACS measurements. This graph
clearly shows that longer incubation times increases the
number of binding sites for a (fluorescent) complementary
primer, thus demonstrating successful RCA on the cell
membrane.
Overview
To test if Rolling Circle Amplification works on cells, cells expressing COMPx were grown according to protocol. These were reacted for 1 hour at 4 °C and 500 rpm with the DBCO coupled primer. These were then spin purified and washed with PBS to remove any remaining primer. One sample was stored for use a control. Three remaining samples were incubated with circular, template, 5 mM DNTP-mix, Phi 29 polymerase and commercially supplied reaction buffer. Sample 1 was allowed to react for one hour before aphidicolin was added to stop the Phi29 polymerase, sample 2 for two and sample 3 for four hours. These samples were then again spin purified and washed with PBS.
All four samples and a blank without clicked DNA were incubated with a fluorescent complementary probe for 1 hour at 4 °C and 500 rpm. These were then spinned down to remove any unbound fluorescent probe. Samples were then resuspended in PBS and ran through the FACS. Figure 1 shows the median fluorescence of the samples. The rise in fluorescence for longer amplification times indicates that Rolling Circle Amplification has been successfully carried out on the cell membrane of our cells.
of incubating for Rolling Circle Amplification. This graph clearly
shows an increase in particle size, this shows that indeed
RCA results in long strands on the membrane.
FACS can also be used to measure particle size. This feature was used to see if the Rolling Circle Amplification actually generated long pieces of DNA or a lot of short sequences were created which gave rise to the graph seen in figure 1. The data from the forward scatter gate was used to generate the graph in Figure 2, the mean forward scatter has been plotted for each sample. This clearly shows that the particle size increases with longer reaction times. Thus demonstrating that, indeed, longer strands were created instead of multiple short fragments.
For a complete protocol for this step click here.
