Biomod/2013/Tianjin/Experiments & Results/Cleavage
optimize the 1st stem-loop structure & terminationCleavage
optimize the 2nd stem-loop structureDelivery device
After we building the track ,we must see that if that can be cleaved by the DNAzyme, which is known as the key part of the track. In fact, this is the reason why we choose the walker with the mechanism of DNAzyme, it's easy to be characterized. We used the DNAzyme to cut the our polymer, and run a denaturation page to separate the DNA strands to see if any of them has been cut to be shorter.
At first we still set the second stem domain as 7bp. After do the polymerizing reaction, we introduce the DNAzyme into the system, also the co-factor. After performing the reaction, we run a PAGE. The double bands in both lane2 and 3 show that there’s a leakage. For the lane2 or 3, the bands means: the origin DNA, the DNA after cleavage, the DNAzyme.(In the order from the upper one) It means that the DNAzyme could cut the monomer, even it is not polymerized. This may cause the low efficiency of the walker.
So we add the length of the 2nd stem to 15bp. And the result turns out to be better. We run the gel for enough long time to separate the 2 bands, which make the band of DNAzyme can’t be seen.
We also detect the concentration of the cleaved and not cleaved DNA, using the florescence integration technique, the result shows that the cleaved DNA strand’s concentration is more than two folds of the DNAzymes’, which means that the multiple cleavage is happened indeed.