Part:BBa_K4245113:Experience

Applications of BBa_K4245113

Lambert_GA 2022 This part was experimented with as a part of the composite part, the hsa-mir-451a RCA Padlock Probe. (BBa_K4245209). RCA with BBa_K4245208 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 451a 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.

Figure 2. Fluorescent reading of RCP produced by miR-451a padlock probe (generated via Probebuilder). BHQ-1 and FAM probes compared to solely FAM probes.

Figure 2 displays a significant decrease in the fluorescence intensity of a triplicate with FAM Probe, BHQ Probe, and the RCP produced as compared to a triplicate of just FAM tagged Probes. This finding experimentally validates the use of ProbeBuilder as a means of producing effective padlock probes.

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