A series of time gradients are set up, and the supernatants of the magnetic beads LABC assembly are respectively taken for PAGE gel electrophoresis.
Design a T sequence with base mutations for qualitative testing. Furthermore, relying on big data, neural network analysis and other bioinformatics tools to design and verify the potential mutation site sequence of the new coronavirus.
Modify the fluorescence of the sequence and use a microplate reader to measure the fluorescence intensity to characterize the sequence content.
During the reaction, the B chain will fall off spontaneously and form a dissociation equilibrium, but this equilibrium is very weak and almost covered by the main reaction. Because F will be added in excess in the main reaction, it is difficult to directly measure the consumption rate of F and it is difficult to accurately quantify it, so the generation rate of CF complex can be used instead. For example, when a fluorescent group is mounted on the 3'end of the C chain, when C is stably bound to A, the fluorescent group is affected by the magnetic field of the magnetic beads and the fluorescence is quenched. Once F replaces C into the solution and moves away from the magnetic beads, it is quenched. The function is invalid, the fluorescent signal is visible, and real-time monitoring is performed, which saves time and effort. (Figure 5)
3.1 Quantitative optimization of magnetic beads AL assembly ratio and absolute dosage
Based on the modeling work, it can be known that the upper and lower limits of the ratio of A to L and the absolute dosage. Based on this, design the assembled magnetic beads with different AL ratios for verification.
3.2 Determine the parameters of the reaction kinetic equation and fit the kinetic equation to provide guidance for the optimization of the reaction time.
Observed from the experimental results, the reaction system itself has background noise, which will affect the fluorescence signal during the qualitative verification and quantitative optimization of molecular experiments, and will affect the current measurement on the DMF equipment. In order to reduce the generation of background noise, we propose the following solutions.
4.1 Change the reagent loading order.
4.2 Design the W chain to form a hairpin secondary structure when it is free in solution.
The sequence used in the experiment is: F→W→T, but the noise has not been significantly improved. Based on the modeling ideas of the DW dynamics part, we analyze and believe that the autonomous dissociation balance of the B chain can be affected when W is not fixed, so that The dissociation balance of B shifts to the right, while W is not fixed and free in solution, it will produce hairpin secondary structure by itself, occupying the complementary sequence between Toehold and not having strand displacement reaction with B.
Based on this, we believe that it is necessary to fix W on the magnetic beads first, replace the B chain, and then add the F chain, namely T→W→F. T preferentially binds to L to fix W, and W is complementary to T before forming a secondary structure to mask the background noise. W replaces B under the restriction of the domain, resulting in two double-strands separated by Toehold2. The base pairing numbers of these two double-strands are both less than the base pairing number of the CF complex. That is, F can efficiently replace the C chain.
Based on the initial experiment, we made some improvements in some parts. Firstly, we found that the SNR(signal to noise ratio) of the electrode was too low because of the too large test electrode, so we made the new plates with smaller test electrodes again and conducted a comparative experiment.
Furthermore, in order to improve the modification efficiency of the chain, we tried the “freezing and thawing” method to modify the chain.
We made two different kinds of electrode plates, one of which had a bigger detection electrode. Nevertheless, since its SNR turned out to be too low in the preliminary experiments, we made another electrode plate with a smaller detection electrode. Both the electrodes are shown in the figure.
We performed the modification- and cut- experiment on both electrodes under the circumstances of the same rinsing method and the same time to inoculate line G, which was 2 hours. Having used the same concentration of Cas9 nickase and sgRNA to cut, we obtained the results indicating that SNR of the signal which came from the electrode with a smaller detection electrode, got lower manifestly. However, the initial signals appeared to be very low on the electrode plate which had a small detection electrode, the detection effect will not be influenced, because it is the degree of the change that matters but not the value of the change. In brief, this kind of electrode plate is the better choice. To demonstrate that the initial signal of this electrode plate could have obvious and effective variations after the possibility of incision has been testified, we performed more experiments, in which the concentration of the sgRNA is higher.
Furthermore, we also compared the cut effect of these two kinds of electrode plates. The result was shown as follows.
Some literature has shown that the modification time of the nucleic acid chain on gold nanoparticles can be shortened by repeated freezing and thawing, and chains can be modified better within only two hours. Therefore, we tried to put the electrode plate with the dripping of the line G solution into the refrigerator at -80 °C. Every 30 minutes, we took out part of the electrode plate to thaw for 5 minutes, and detected the electrical signal, until the modification time reached 4 hours. However, our detection results show that the chain can’t be modified very well, which is even worse than the long-term modification. This may be based on the fact that the specific surface area of the detection electrode is smaller than that of gold nanoparticles. In the future, we will consider increasing the modification concentration of line G and applying repeated freeze-thaw-method to improve the efficiency of chain modification.