Difference between revisions of "Part:BBa K4245006:Experience"

Revision as of 10:57, 11 October 2022

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Lambert_GA 2022 Rolling Circle Transcription (RCA) was successful with this part. The products of RCA are long DNA strands composed of repeating complementary sequences of the used padlock probe. Therefore, one way in which the success of RCA can be determined is by running the rolling circle products (RCP) on an agarose gel. Since a fluorescent band very close to the wells would indicate the presence of an extremely long DNA strand, our RCP was run on a gel. The result was a really long DNA strand close to the well.

Figure 1. Image of gel ran with miRNA-1 RCP product.

By analyzing the results on the gel, our team concluded that a very long strand of DNA, likely the RCP, was produced. The gel exhibited a fluorescent band of DNA very close to the well, which indicates that a long strand of DNA, greater than 1 kB, was produced due to our reaction (see Fig. 1). As a result, we can infer that the RCA reaction allowed the creation of a really long DNA stand -- our RCP.
The RCP was also tested with the split lettuce aptamer. DFHBI-1T and the lettuce right and the modified lettuce left was added to the RCP, and the fluorescence was read on the plate reader.

Figure 2. Graph of split Lettuce reaction with RCP. The values represent the change in fluorescence before and after the reaction with DFHBI-1T took place.

As seen in Figure 2, the increase in fluorescence of the RCP + Lettuce + dye was significantly greater than the controls, which suggests that the split Lettuce was successfully bound to the RCP. In addition, the DFHBI-1T was also successfully bound within the split lettuce secondary folding. According to these results, RCA was successful, and the reaction between the split lettuce and RCP was successful as well.
In addition to split lettuce, RCA and RCT were also tested with the FAM and BHQ1 labeled linear probes.
As shown by Figure 3, there is a statistically significant decrease in the fluorescent output of a triplicate with FAM Probe, BHQ Probe, and RCP as compared to a triplicate of just FAM tagged Probes. This confirms that we did produce our desired RCP in the RCA reaction and that this mechanism was an effective reporting method for our sensor.

Figure 3. Fluorescent Read of Rolling Circle Product for miRNA-133-3p and miRNA-1-3p

Therefore, RCA created RCP that can be quantified by our chosen reporting mechanisms.

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