Difference between revisions of "Part:BBa K4245009:Experience"

Latest revision as of 13:00, 12 October 2023

Applications of BBa_K4245009

Taken from BBa_K4245204
Lambert_GA 2022
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.
The RCP was also tested with the FAM and BHQ1 tagged linear DNA probes.
As shown by Figure 2, 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 2. 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 mechanism.

Lambert_GA 2023
Specificity Testing

While Lambert iGEM has been utilizing rolling circle amplification (RCA) to detect a single isolated microRNA (miRNA), human blood serum contains a total of 204 detectable miRNAs (Wang et al., 2012). Research conducted by Jonstrup et al. in 2006 found that the padlock probe ligates on a perfectly matching RNA template, distinguishing between differences in the target and other sequences. To test whether padlock probes would be able to detect specific miRNA, and therefore be applicable for serum testing, we ran RCA using the hsa-miR-1-3p padlock BBa_K4245200 in the presence of four different miRNA sequences (see Fig. 1). The first is the original miR-1 sequence ,BBa_K4245006, which is expected to hybridize to the padlock and result in the greatest fluorescence decrease. Two sequences with differing single nucleotide variants (SNVs) found from the National Library of Medicine microRNA 1-1 database were utilized to determine the specificity of RCA: one with a single SNV, BBa_K4683003, and one with three SNVs, BBa_K4683004. hsa-miR-133a-3p, BBa_K4245009, was also included to ensure the padlock would not ligate to any miRNA.
Figure 1. Image of gel ran with miRNA-1 RCP product; A: eRCA with 40.8 pM miR-1; B: negative control (no enzymes)

Figure 1. Comparison of sequences used to test specificity of hsa-miR-1-3p RCA padlock: 1 bp SNV in the seed region, 3 SNVs outside of seed, and miR-133a-3p

We ran the reactions and control on a gel electrophoresis; only the well with 40.8 pM of miR-1 showed visible bands of DNA near the top of the wells, which is likely our RCP (see Fig. 2). We then tested the RCP with linear DNA probes and quantified the resultant fluorescence in a plate reader (see Fig. 3) The RCA reaction utilizing the miR-1 padlock probe with miR-1 exhibited significantly less fluorescence than the other miRNAs. Since linear DNA probes produce a negative correlation between fluorescence and miRNA concentration, this result, along with the gel, indicates that RCA is specific to single nucleotide differences. Figure 1. Image of gel ran with miRNA-1 RCP product; A: eRCA with 40.8 pM miR-1; B: negative control (no enzymes)
Figure 2. Gel results: A: miR-1, B: 1 SNV, C: 3 SNVs, D: miR-133a- Image of an agarose gel run with RCP from RCA run with 40.8 pM of each miRNA in reaction.
Figure 3. Comparison of RCA with miR-1, 1SNV, 3SNVs, and 133a fluorescence output using linear DNA probes

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UNIQ80991bed05f73734-partinfo-00000009-QINU UNIQ80991bed05f73734-partinfo-0000000A-QINU

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 <b> Lambert_GA 2023</b>
 <br>
-Specificity Testing
+Specificity Testing<br>
 	While Lambert iGEM has been utilizing rolling circle amplification (RCA) to detect a single isolated microRNA (miRNA), human blood serum contains a total of 204 detectable miRNAs (Wang et al., 2012). Research conducted by Jonstrup et al. in 2006 found that the padlock probe ligates on a perfectly matching RNA template, distinguishing between differences in the target and other sequences. To test whether padlock probes would be able to detect specific miRNA, and therefore be applicable for serum testing, we ran RCA using the hsa-miR-1-3p padlock  <partinfo>BBa_K4245200 </partinfo> in the presence of four different miRNA sequences (see Fig. 1). The first is the original miR-1 sequence ,<partinfo>BBa_K4245006 </partinfo>, which is expected to hybridize to the padlock and result in the greatest fluorescence decrease. Two sequences with differing single nucleotide variants (SNVs) found from the National Library of Medicine microRNA 1-1 database were utilized to determine the specificity of RCA: one with a single SNV,  <partinfo>BBa_K4683003 </partinfo>, and one with three SNVs,  <partinfo>BBa_K4683004 </partinfo>.  hsa-miR-133a-3p, <partinfo>BBa_K4245009 </partinfo>, was also included to ensure the padlock would not ligate to any miRNA.
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