RNA

Part:BBa_K4683004:Experience

Designed by: Manasvi Gupta, Natalie Tan, Vandana Vijay Kuma, Kate Sharer   Group: iGEM23_Lambert-GA   (2023-10-10)


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Applications of BBa_K4683004

Lambert_GA 2022
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 1. Image of gel ran with miRNA-1 RCP product; A: eRCA with 40.8 pM miR-1; B: negative control (no enzymes)
Figure 3. Comparison of RCA with miR-1, 1SNV, 3SNVs, and 133a fluorescence output using linear DNA probes

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