Multistep-SDA


Overview

Strand Displacement Amplification (SDA) is a method to produce a large amount of ssDNA by using DNA polymerase with strand displacement activity and nickase, which recognizes dsDNA and puts a nick on one of the strands. We focused on the system 1 developed by Dr. Komiya at JAMSTEC. This system is a multi-step combination of SDA reactions to amplify ssDNA. The SDA reaction is originally subject to amplification even in negative control, but in this system, negative control is suppressed by various innovations. We call this system “multistep-SDA" and conducted experiments.
For more information about the principle of multistep-SDA, see Proposed Implementation_Amplification
For more information about the actual experimental procedure of multistep-SDA, see Experiments_Multistep-SDA

1. Preliminary Experiments

Purpose:

We attempted to confirm the amplification from single stranded nucleic acid by the mechanism described in the paper.
The paper uses NEBuffer 2 as the buffer, but we used NEBuffer 2.1. The only difference between NEBuffer 2 and NEBuffer 2.1 is whether DTT or albumin is used in the buffer, which does not seem to affect the enzyme activity 2. In addition, in the paper, the 3' ends of the template DNA strands are chemically modified with carboxytetramethylrhodamine (TAMRA). We used ssDNA without this modification as the template. Fluorescence intensity was measured using MB as in the paper.

Result:

The fluorescence changes were plotted below.

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Consideration:

It could be distinguished from NC if the target concentration was at least 100 pM. In the paper, no amplification of NC was observed after 150 min, but in this experiment, amplification started at about 25 min.
We conducted further experiments to elucidate the cause of the apparently faster amplification than in the paper.

2. Pursuing the Cause of Amplification in NC

Purpose:

We wanted to clarify the cause of the apparent faster amplification than in the paper. To determine which step was responsible, we performed experiments with each step of multistep-SDA, as well as with two or three steps of multistep-SDA connected together. Since the MB used in the paper cannot detect the amplification products of the first and second steps of SDA, we also measured the fluorescence intensity using SYBR Green I or SYBR Green II. Additionally, we performed experiments using the optimized buffer in the paper (we call it LT Buffer) instead of NEBuffer 2.1.

Result:

The fluorescence changes were plotted below.

Step 1

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Figure 1: Step 1

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Step 2

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Figure 2: Step 2

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Step 3

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Figure 3: Step 3

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Step 12

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Figure 4: Step 12

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Step 23

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Figure 5: Step 23

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Step 123

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Figure 6: Step 123

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Consideration:

When only step1 and step2 were used, the amplification speed was slow and amplification of NC was not observed, but amplification of target was also almost unobservable. It was confirmed that amplification efficiency increased with the number of steps, but when two or more steps were linked, amplification of NC was observed. Also, when NEBuffer 2.1 and LT Buffer were compared in step123, no significant difference was observed.

Consult with Extra Adviser Dr. Komiya

In the paper, no amplification of NCs was observed in the 3step-SDA reaction, but in our experiment, amplification of NCs was observed by connecting multiple steps of SDA. We asked Dr. Komiya, the author of the paper and our extra adviser, for tips. He told us that it is important that the 3' ends of template DNA are chemically modified with carboxytetramethylrhodamine (TAMRA) .

3. Follow-up Experiment with TAMRA Modified Template DNA Strands

Purpose:

Based on the advice, we performed follow-up experiments with template DNA strands whose 3' ends are chemically modified with TAMRA. Fluorescence intensity was measured using MB like in the paper.

Result:

Within 200 min of observation, amplification was observed at target concentrations of 10 nM or higher for only step3 and step23, and at target concentrations of 100 pM or higher for step123. In all cases, NC amplification was suppressed.

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Consideration:

By using a template with TAMRA modification at the 3' end, amplification was confirmed within 200 min of the observation when the target concentration was 10 nM or higher for only step3 and step23. For step 123, amplification was confirmed when the target concentration was 100 pM or higher. In all cases, amplification of NCs was not confirmed during the observation time.

Conclusion

By using a template with TAMRA modification at the 3' end to link multiple stages of SDA, it was confirmed that target amplification could be efficiently achieved while NC amplification was suppressed. The increase in amplification efficiency by increasing the number of stages was also confirmed. However, amplification was observed within 200 min only when the target concentration was 100 pM or higher, and this mechanism cannot be used in POIROT, which aims for detection in the fM order. Therefore, Multistep-SDA must be combined with other amplification mechanisms.

Multistep-SDA is a system that uses short nucleic acids as input, and in principle, any sequence of nucleic acids can be amplified by changing the template sequence. In addition, this system is characterized by the fact that it proceeds at 37 ºC without the need for temperature changes. Furthermore, it is also possible to amplify dsDNA by changing the template sequence and eliminating the nicking site. Therefore, multistep-SDA is a useful system that can be easily connected to other amplification methods that proceed at 37 °C and to the CRISPR-Cas system that targets dsDNA.

References


  1. Komiya, K., Noda, C. & Yamamura, M. (2024). Characterization of Cascaded DNA Generation Reaction for Amplifying DNA Signal.New Gener. Comput. 42, 237-252. https://doi.org/10.1007/s00354-024-00249-2

  2. New England Biolabs. (2013). Why did you remove DTT from your restriction enzyme buffers? https://www.neb.com/ja-jp/faqs/2013/02/28/why-did-you-remove-dtt-from-your-restriction-enzyme-buffers