ThisAmp is a modified version of the SDA reaction that incorporates the TWJ structure, and it was reported by Lee et al. 1.
For more information about the principle of TWJ and SDA, see Proposed Implementation_Amplification.
For more information about the actual experimental procedure of ThisAmp, see Experiments_ThisAmp.
This experiment was conducted to determine whether the annealing process of target, template, and helper to form a three-strand complex, which is done in the prior study, is truly necessary. If the annealing step can be omitted, the reaction could begin with a simple mixture of these DNAs and enzymes, making the mechanism more accessible and convenient for use in household settings.
The fluorescence changes were plotted below.
In the paper with annealing operation, the amplification curves for target 1 fM and negative control (NC) were distinguishable. However, skipping the annealing step resulted in distinguishable amplification curves only up to target concentrations of 100 nM, which is approximately \(10^8\) orders of magnitude worse than the results presented in the original paper. Based on these results, it was concluded that the annealing process is necessary.
We attempted to verify the amplification mechanism described in the paper. While the paper used NEBuffer 3.1 as the buffer, we opted to use NEBuffer r3.1. The only difference between these buffers is that albumin in the buffers is recombinant in NEBuffer r3.1. Additionally, the paper employed vent (exo-) DNA polymerase, but we used Bst 2.0, which had already demonstrated amplification in EXPAR. To accommodate Bst 2.0, we replaced the ThermoPol Reaction Buffer with Isothermal Amplification Buffer.
The fluorescence changes obtained are shown in the graphs below. The left graph represents the results up to 60 min. While we conducted measurements at 60 min, an increase in fluorescence intensity was observed in less than 2 min. The right graph zooms in on the first 10 min of the reaction.
The amplification curves for 10 nM and NC can be distinguished; however, the difference in the starting time of amplification is less than 2 min. This means that with even a slight extension of the reaction time, it becomes difficult to differentiate between the two. This could lead to an increase in false positive rates when used in a home setting. Therefore, this issue needs to be addressed for more reliable detection.
To suppress the amplification of NC and increase the time gap between the start of amplification for the target and NC, we performed tuning of the template concentration.
The fluorescence changes were plotted below.
When the template concentration was lowered, it was observed that the amplification efficiency decreased, and at concentrations lower than 1/64 of the value used in the reference paper, no amplification could be detected within 60 min in SYBR Green Ⅰ fluorescence measurement. However, lowering the template concentration would not only slow the amplification speed but also reduce the amount of amplified product. Given the goal of amplifying and quantifying nucleic acids, lowering the template concentration was not desirable. Therefore, the template concentration was kept at 50 nM, as specified in the original paper.
Tuning of polymerase concentration was performed to suppress NC amplification by decreasing the rate of amplification while maintaining the amount of amplified product, and to increase the difference between the time when target amplification begins and the time when NC amplification begins.
Fluorescence intensity was as shown in the left figure, and for polymerase concentration, the time until fluorescence intensity reached \(8 \time 10^5\) is shown in the right figure. Error bars represent standard deviation.
The largest difference in the time required for the fluorescence intensity to reach \( 8 \times 10^5 \) between the target concentration of 100 pM and 0 M was observed when the polymerase concentration was 1/4 of the published data, 0.012 U/µL. The difference at this time was about 20 min. Based on this, we thought thatwe could solve the problem described in 2. by reducing the polymerase final concentration to 0.012 U/µL.
To confirm the LoD of this system, the experiment was repeated with the conditions obtained in section 4. and 5.
The fluorescence changes were plotted below.
The LoD was 10 nM in 2. before tuning, but after tuning, the LoD was 100 pM.
ThisAmp had a LoD of 100 pM after optimization. This reaction is characterized by the fact that it proceeds at a constant 55 ℃ and requires no temperature change. ThisAmp is useful in that it can form a TWJ and amplify miRNAs, but it is not suitable for miRNA amplification due to its long target length (59 mer) . In addition, since the target, template, and helper must be annealed before adding the enzyme, it is difficult to connect other amplification mechanisms before this reaction considering a one-pot amplification system. Furthermore, since the main amplification product is dsDNA, it would also be difficult to connect other amplification mechanisms behind this reaction.
Lee, S., Jang, H., Kim, H. Y., & Park, H. G. (2020). Three-way junction-induced isothermal amplification for nucleic acid detection. Biosensors and Bioelectronics, 147, 111762. https://doi.org/10.1016/j.bios.2019.111762