Difference between revisions of "Part:BBa K4683013:Design"
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<partinfo>BBa_K4683013 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4683013 SequenceAndFeatures</partinfo>
  
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===Design Notes===
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Design Notes
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The sequence of hsa-mir-150-5p was taken from miRBase, and we chose the mature sequence as it is found in circulation. No changes were made as these were the target sequence.
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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).
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<html><img src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/group-1-21.png
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alt="Figure 1. Image of gel ran with miRNA-1 RCP product; A: eRCA with 40.8 pM miR-1; B: negative control (no enzymes)
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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  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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<b> References </b>
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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
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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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===Source===
 
===Source===
  
 
Mirbase entry: HSA-mir-150-5P. miR. (n.d.). https://mirbase.org/mature/MIMAT0000451
 
Mirbase entry: HSA-mir-150-5P. miR. (n.d.). https://mirbase.org/mature/MIMAT0000451

Revision as of 04:47, 12 October 2023


hsa-mir-122-5p


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Design Notes

The sequence of hsa-mir-150-5p was taken from miRBase, and we chose the mature sequence as it is found in circulation. No changes were made as these were the target sequence.

Source

Mirbase entry: HSA-mir-150-5P. miR. (n.d.). https://mirbase.org/mature/MIMAT0000451