EXPAR


Overview

Exponential amplification reaction (EXPAR) is a reaction in which SDA occurs exponentially. After receiving miRNA or ssDNA as input and hybridizing them with template ssDNA, DNA polymerase with strand displacement activity extends to the 5' end of the template to form dsDNA. The restriction enzyme recognition site on the dsDNA is nicked by nickase, and the polymerase recruits to the nicking site to cause strand displacement amplification. If the sequence of ssDNA of the amplified product is the same as the sequence of the input signal, the amplified product plays the same role as the target, and exponential amplification occurs. A 50 ℃ reaction is possible by using Bst 2.0 and Nt.BstNBI. We experimented with existing methods 1.
For more information about the principle of SDA, see Proposed Implementation_Amplification.
For more information about the actual experimental procedure of EXPAR, see Experiments_EXPAR.

We conducted each experiment in tripricate.

1. Preliminary Experiment

Purpose:

We attempted to confirm the amplification in the mechanism of the paper. The paper used dsGreen, but we used SYBR Green Ⅰ. Since SYBR Green Ⅰ is a double-stranded DNA staining reagent with the same performance as dsGreen 2, this change does not affect the experimental results. In addition, OPC-purified oligo DNA was used.

Result:

The fluorescence changes were plotted below.

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Consideration:

As in the paper, we were able to distinguish between the amplification curves for target 1 pM and 0 M. The amplification curve for target 1 pM started to rise about 7 min after the start of the reaction, which is consistent with the paper.

2. Heat Inactivation of Nickase

Purpose:

The reaction temperature of EXPAR is 50 ℃. In designing the ODE model of EXPAR in the Dry Lab, it was necessary to consider thermal inactivation of nickase during the reaction. Therefore, we conducted an experiment to confirm the thermal inactivation of nickase in the Wet Lab. Using the protocol in the ODE model paper 3 as a reference, we performed amplification reactions as usual and measured changes in fluorescence intensity using pre-incubated nickase solution for a certain period of time. The conditions other than pre-incubation of the nickase were the same as in the preliminary experiment.
For more information about the ODE model, see Model.

Result:

The fluorescence changes were plotted below. Nickase was not added in NC.

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Consideration:

No change in the amplification curve due to the pre-incubation time of the nickase was observed. It can be considered that changes in the enzymatic activity of the nickase due to reaction time are negligible.

3. Comparison of Purification Level

Purpose:

We purchased OPC-purified oligo DNA and PAGE-purified oligo DNA. We performed an experiment to see if the difference in amplification efficiency between these two purification methods makes a difference.

Result:

The left graph shows the result with OPC-purified oligo DNA and the right graph shows the result with PAGE-purified oligo DNA.

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Consideration:

The fluorescence intensity was higher when PAGE-purified oligo DNA was used; since PAGE purification is a method that yields highly purified oligo DNA 4, the higher fluorescence intensity could be attributed to the higher concentration of the oligo DNA of interest in the same volume of solution. As for the amplification speed, there was almost no difference depending on the purification method.

When OPC-purified oligo DNA was used, the experimental procedure was the same as in the 1. Preliminary Experiment, but the amplification speed was about five times faster than in the 1.

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The left graph shows the fluorescence change in 1. and the right graph shows the fluorescence change when OPC-purified oligo DNA was used in 3. After rearranging the conditions, it was found that Bst 2.0, a polymerase, was purchased again between section 2. and 3., and that only the lot of Bst 2.0 was different between section 1. and 3. Therefore, we concluded that the difference in amplification speed between the two was due to the difference in Bst 2.0 lot. In subsequent experiments, we used the new Bst 2.0 lot and re-examine the response to template / nickase / polymerace concentration.

4. Tuning of Template Concentration

Purpose:

An attempt was made to further lower the LoD by tuning the template concentration.

Result:

The fluorescence changes were plotted below.

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Consideration:

The higher the template concentration, the stronger the fluorescence intensity. The overall amplification speed was so fast that differences in amplification speed due to differences in template concentration were buried, and differences in amplification speed could not be measured. Since lowering the template concentration decreases the amount of amplified product, it is desirable not to lower the template concentration for the purpose of amplifying and quantifying nucleic acids. Therefore, in subsequent experiments, template concentration was set at 25 nM as in the paper.

5. Tuning of Nickase Concentration

Purpose:

Attempts were made to further lower the LoD by tuning the nickase concentration.

Result:
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Consideration:

The largest difference in the time for the amplification curve to rise between the target concentration of 100 pM and 0 M was observed when the nickase concentration was 1/2 times of that in the paper (0.125 U/µL). We considered the possibility of increasing the difference in the time until the amplification curve rises between 100 pM and 0 M by further decreasing the nickase concentration.

6. Tuning of Polymerase Concentration

Purpose:

We attempted to further lower the LoD by tuning the polymerase concentration. Nickase concentration was set to 1/2 of the published value (0.125 U/µL).

Result:

The fluorescence changes were plotted below.

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Consideration:

The difference in the time to amplification curve between target concentration 100 pM and 0 M was greatest when the polymerase concentration was 1/4 of the published value (0.02 U/µL). We considered the possibility of increasing the difference in the time to amplification curve rise between the 100 pM and 0 M by further lowering the polymerase concentration.

7. Tuning of Polymerase and Nickase Concentration

Purpose:

We attempted to further lower the LoD by simultaneously tuning the polymerase and nickase concentrations.

Result:
Fluorescence changes were obtained as shown in the graph below.

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Consideration:

When the polymerase concentration was 1/16 times of that in the paper (0.005 U/µL) and the nickase concentration was 1/8 times of that in the paper (0.0625 U/µL), the amplification curves at 1 pM and 0 M could be distinguished.

8. Re-experiment under Tuned Conditions

Purpose:

To observe the LoD of this system, the experiment was repeated with a lower target concentration using the conditioning obtained in section 7.

Result:

The fluorescence changes were plotted below.

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Consideration:

LoD was 1 pM.

Conclusion

The LoD was 1 pM when the new lot of Bst 2.0 was used with a polymerase concentration of 0.005 U/µL and nickase concentration of 0.0625 U/µL. EXPAR is an efficient amplification system and is promising to achieve the required amplification efficiency for POIROT within 30 min. On the other hand, EXPAR was shown to be unstable and highly dependent on the lot of the enzyme. The inclusion of EXPAR in the amplification system would reduce the overall robustness of the system. So we were forced to consider a different amplification system.

References


  1. Carter, J. G., Orueta Iturbe, L., Duprey, J. H. A., Carter, I. R., Southern, C. D., Rana, M., Whalley, C. M., Bosworth, A., Beggs, A. D., Hicks, M. R., & Tucker, J. H. R. (2021). Ultrarapid detection of SARS-CoV-2 RNA using a reverse transcription-free exponential amplification reaction, RTF-EXPAR. Proceedings of the National Academy of Sciences, 118(35), e2100347118. https://doi.org/10.1073/pnas.2100347118

  2. Funakoshi Co., Ltd. (n.d.). dsGreen: Kakusan geru senshoku shiyaku / riaru taimu PCR-yo SYBR®-kei keikō shikiso. https://www.funakoshi.co.jp/contents/67539

  3. Özay, B., Murphy, S. D., Stopps, E. E., Gedeon, T., & McCalla, S. E. (2022). Positive feedback drives a secondary nonlinear product burst during a biphasic DNA amplification reaction. Analyst, 147(20), 4450-4461. https://doi.org/10.1039/D2AN01067D

  4. FASMAC. (n.d.). DNA/RNA jutaku gosei seisei guredo no sentaku ni tsuite. https://fasmac.co.jp/dna_rna_purify_grade