DNA

Part:BBa_K4245103:Experience

Designed by: Akshaya Poonepalle, Michelle Jing, Shivaek Venkateswaran, Daeun Lee, Sahana Ram Narayanan, Richard Jiang, Sishnukeshav Balamurali, Christina Yi, Varnica Basavaraj, Manaswi Gorle, Janet Standeven   Group: iGEM22_Lambert_GA   (2022-09-22)
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Applications of BBa_K4245204

Rolling Circle Amplification(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 133a RCP product.RCP is visible in wells 7 and 8.


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.


Linear Probes with RCP
We use linear probes as a means to quantify and report the miRNAs that we sensed through rolling circle amplification (RCA) reactions. To go beyond verifying that linear probes are efficient means to do the aforementioned tasks through testing with complement of the linear probes, we wanted to confirm that they are able to quantify the miRNAs experimentally (see RCA Protocols)

Figure 2. Fluorescent Read of Rolling Circle Product for miRNA-133-3p and miRNA-1-3p

As shown by Figure 2, there is 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 for our miRNA-1-3p and miRNA-133a-3p sensors and that this mechanism was an effective reporting method for our sensor.
Therefore, RCA created RCP that can be quantified by our chosen reporting mechanism.


User Reviews

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