Part:BBa_K4667003
H1 H2 are primers for the isothermal amplification process
H1
Initiating the HCR amplification reaction
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 9
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Electropherogram of the results of miRNA-initiated and HCR isothermal amplification reaction.lane 1： H1(1 µM)；lane 2： H2(1 µM)；lane 3：1 µM H1+ 1 µM H2； lane 4：1 µM H1+ 1 µM H2+ 50 nM miR； lane 5：1 µM H1+ 1 µM H2+ 100 nM miR； lane 6：1 µM H1+ 1 µM H2+ 200 nM miR； lane 7：1 µM H1+ 1 µM H2+ 500 nM miR-； lane 8：1 µM H1+ 1 µM H2+ 1000 nM miR
Electrophoretic graph of the results of HCR isothermal amplification reaction triggered by miRNA, only H1 H2 cannot trigger the amplification reaction, but it can be triggered by the addition of miRNA and the shapes of the amplification bands triggered by different concentrations of miRNA are similar, and all of them show the results of trapezoidal bands, but the higher the concentration of miRNA, the faster the reaction speed.
Specificity results of miRNA-initiated isothermal amplification reaction
As can be learned from the figure, the method we have developed is characterized by high specificity. Different miRNAs can target different hairpin structures, thus triggering an amplification reaction, which occurs with a clear difference between the color change and the blank as well as other kinds of miRNAs, demonstrating good method specificity. After the HCR amplification reaction, different miRNA amplification products produce different degrees of signals after binding to the Cas12a protein. cas12a protein and CrRNA can recognize and cleave miRNA amplification products to generate signals.
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