We started the experiment of DNA Walker at the end of September and independently designed the assembly procedure of magnetic beads and the reaction procedure of chain replacement. Our experimental results on October 9th show that DNA Walker can transform the input line T signal into the output C:F double-chain signal under a certain amount of line T and line W concentration and through appropriate reaction procedures. However, our experimental results are not perfect. In the experimental results on October 9th, we found that the positive control had obvious C:F double-stranded detection and the negative control also had weak C:F double-stranded signal, which made us notice the machine leakage of DNA Walker. Therefore, in the subsequent experiments, we hope to reduce the impact of leakage by reasonably designing the reaction program.
We verified the lineG cutting in PCR tube and on electrode respectively. Our experimental results show that, when the reaction occurs in an PCR tube, the C:G double strand can be cut by cas9 nickase, and some short segments are produced and can be shown in PAGE. We tried other reaction conditions, such as cutting only with lineF or lineG, and they could not produce such a cutting band. This shows that the cutting of C:G is specific, which is consistent with the design.
We also carried out verification experiments on the electrode. We found that when there was line C in the system, cutting would occur on the electrode and the chemical current would decrease, which was completely different from negative control and blank control. This shows that the cutting of C:G is also specialized when it is on the electrode, and it can indeed produce considerable chemical current changes. Of course, we have also noticed some problems such as surfactant and unstable electrode modification, and we will improve these parts in subsequent experiments.
DNA Walker outputs C:F double strand to E-CRISPR, while E-CRISPR only needs lineC, which makes lineG need to compete with F chain. We also verified this part on the PCR tube and electrode respectively, and the results show that the G chain can compete with the C chain both in the PCR tube and electrode. Of course, at present, this kind of competition needs to be promoted by providing a certain amount of heat. We plan to make this part more efficient by prolonging the reaction time or changing the experimental design in the later stage.