Difference between revisions of "Part:BBa K4683006:Design"

Latest revision as of 04:26, 12 October 2023

Design Notes
For the padlock probe (PLP) design, part of the reverse complement of the miRNA makes up each end of the padlock probe. To determine where the reverse complement is split properly, we determined the annealing temperatures of each arm through SnapGene. To allow successful hybridization and maximize the binding efficiency of the miRNA and the padlock arms, the arms need to have the same annealing temperature. Furthermore, we added a phosphate group modification to the 5’ end of the padlock sequence to allow ligation by SplintR ligase (Jonstrup et al., 2006).
Figure 1. Image of gel ran with miRNA-1 RCP product; A: eRCA with 40.8 pM miR-1; B: negative control (no enzymes)

Additionally, the alignment of the 5’ and 3’ ends is essential to determine where each part of the padlock and its arms matches up to the target miRNA (Liu et al., 2013). The miRNA strand hybridizes antiparallel to the padlock arms Therefore, the 5’ end of the miRNA will end up overlapping the 5’ PLP arm, and the 3’ end of the miRNA will end up overlapping the 3’ PLP arm (Liu et al., 2013).

References
Jonstrup, S. P., Koch, J., & Kjems, J. (2006). A microrna detection system based on padlock probes and rolling circle amplification. RNA, 12(9), 1747–1752. https://doi.org/10.1261/rna.110706

Liu, H., Li, L., Duan, L., Wang, X., Xie, Y., Tong, L., Wang, Q., & Tang, B. (2013). High specific and ultrasensitive isothermal detection of microRNA by padlock probe-based exponential rolling circle amplification. Analytical Chemistry, 85(16), 7941–7947. https://doi.org/10.1021/ac401715k

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-Figure. RCP from RCA reaction with miR-20b, miR-328,miR-146, miR-150, miR-122, and miR-30c; visible bands can be seen near the top of the wells.
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-Additionally, the alignment of the 5’ and 3’ ends is essential to determine where each part of the padlock and its arms matches up to the target miRNA (Liu et al., 2013). The miRNA strand hybridizes antiparallel to the padlock arms (see Fig. 1) Therefore, the 5’ end of the miRNA will end up overlapping the 5’ PLP arm, and the 3’ end of the miRNA will end up overlapping the 3’ PLP arm (Liu et al., 2013).
+Additionally, the alignment of the 5’ and 3’ ends is essential to determine where each part of the padlock and its arms matches up to the target miRNA (Liu et al., 2013). The miRNA strand hybridizes antiparallel to the padlock arms  Therefore, the 5’ end of the miRNA will end up overlapping the 5’ PLP arm, and the 3’ end of the miRNA will end up overlapping the 3’ PLP arm (Liu et al., 2013).
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