Difference between revisions of "Part:BBa K3905015"
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<partinfo>BBa_K3905015 short</partinfo>
 
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Amplification probe required for the isothermal amplification of miRNA using Recombinase polymerase amplification. RPA of the miRNA required the design of the 210-3p Probe 1. Probe 1 binds to the 5' end of the miRNA. This probe is phosphorylated on its 5' end. Through phosphorylation, DNA ligase is able to ligate 210-3p Probe 1 to 210-3p Probe 2. Allowing RPA to occur for the formation of DNA.  
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<h4>This is one of the two probes required for the isothermal amplification of miR-210-3p. </h4>
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For this to occur, the forward primer 5' AAACAAGAAACAGGATAATACGACTC 3' is added, which binds to this probe.
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<h2> How our miRPA works </h2>
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<img width="500px" src="https://2021.igem.org/wiki/images/1/1b/T--City_of_London_UK--RPA2.png">
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As seen in the diagram above, two DNA probes, one with 5’ phosphorylation, bind to the miRNA, and are ligated together by DNA ligase. Then, primers are added, with DNA polymerase, and complementary strands to the ligated probes are synthesised. Then, RPA can take place: primers, associated with recombinase protein so they can dislodge the strands, replicating them in a similar method to PCR, but as no heat cycles are required to break up the strands, the process can take place isothermally.  
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In order for the miRNA to be detected, we decided to use ‘asymmetric RPA’: an excess of forward primers are added (usually 5x the amount), so an excess of the strand that was originally miRNA form, so there is now ssDNA with the miRNA code in DNA. This can be detected by our toehold switches.
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Revision as of 21:26, 21 October 2021


210-3p Probe 1

This is one of the two probes required for the isothermal amplification of miR-210-3p.

How our miRPA works

As seen in the diagram above, two DNA probes, one with 5’ phosphorylation, bind to the miRNA, and are ligated together by DNA ligase. Then, primers are added, with DNA polymerase, and complementary strands to the ligated probes are synthesised. Then, RPA can take place: primers, associated with recombinase protein so they can dislodge the strands, replicating them in a similar method to PCR, but as no heat cycles are required to break up the strands, the process can take place isothermally.


In order for the miRNA to be detected, we decided to use ‘asymmetric RPA’: an excess of forward primers are added (usually 5x the amount), so an excess of the strand that was originally miRNA form, so there is now ssDNA with the miRNA code in DNA. This can be detected by our toehold switches.





Sequence and Features


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]