PROCEEDINGS of the
23rd International Congress on Acoustics
9 to 13 September 2019 in Aachen, Germany

DNA amplification by sound and ultrasound frequency vibration
Ryu Kobayashi1; Seiji Yoneda1; Shigeo Yamaguchi1
1

Kanagawa University, JapanABSTRACT
In the Polymerase Chain Reaction (PCR) method, which is a strong DNA amplification technology, a stepwith a high temperature of 94 ℃ is inevitably included to denature double-stranded DNA to single-stranded
DNA. However, the thermal denaturation has problems causing DNA damage and enzyme deactivation. Toovercome them, we have proposed and developed a new technology for DNA denaturation and amplification,
namely, vibration-driven DNA amplification method (the frequency ranges from 100 Hz to 25 kHz). In ourmethod, DNA is denatured and amplified by vibrating a small plastic tube with at sound to ultrasoundfrequencies using a wave transducer. In the tube, DNA, enzyme, and other materials are mixed and dissolvedin water. Using the DNA amplification system we developed, we examined the optimum condition for DNA
denaturation and amplification by changing amplitude, frequency and time of vibration. Maximum
amplification rates were obtained at frequencies from 100 Hz to 800 Hz. Moreover, through the agarose gelelectrophoresis, we found that no DNA was damaged by vibration.
Keywords: DNA, PCR, Vibration

1. INTRODUCTION
In the DNA amplification technology, the PCR method conventionally used includes a process ofdissociating DNA from double-stranded into single-stranded by high temperature of 94 ° C. However,this heat denaturation has problems such as DNA damage and enzyme deactivation. Therefore, wehave proposed and developed DNA denaturation and amplification method using vibration in the audio
frequency band without using high temperature conditions.
In our method, DNA is denatured by vibrating the DNA solution at audible frequencies using awave transducer. To analyze the results of DNA amplification by vibration, a microchip -type
electrophoresis was used. Furthermore, the energy applied to DNA by vibration was estimated, andthe relationship between the estimated values and the amplification of DNA was discussed.

2. METHOD
2.1 The PCR method
The conventional thermal PCR is a method of amplifying a target DNA about 2 N times byperforming N cycles of a three-step thermal cycle reaction using a primer and an enzyme that bind toboth ends of the DNA at the target site.
The thermal cycle is explained as follows. First, double-stranded DNA is denatured into
single-stranded DNA by applying heat of 94 (denaturation). Second, the temperatures of thesingle-stranded DNA and the primer are lowered to 55 ℃ to mutually complement bind (annealing).
Finally, by raising the temperature to 72 ℃, the enzyme works to extend the primer. These three steps
are one cycle.
Figures 1 and 2 show the DNA model during PCR and, the thermal cycle of the PCR,
respectively.

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r201870084yq@jindai.jp

Figure 1 – DNA model during PCR

Figure 2 – Thermal cycle of PCR
2.2 Vibration-driven PCR
The conventional PCR method has a problem that it damages DNA and enzymes because it uses
high temperature. Therefore, we have proposed and developed a new technology of denaturation andamplification of DNA by vibration in stead of using heat. In the vibration-driven PCR method,
accurate amplification, significant reduction of time, and high efficiency are expected.
The method was performed by placing a wave transducer in a thermostatic chamber set to theactivation temperature (37 ℃) of the enzyme to be used.
Figure 3 shows device configuration of the vibration PCR method.

Figure 3 – Configuration of the vibration-driven PCR method

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The vibration PCR method is performed through two-step process. First, the DNA solution isdenatured by vibration. Next, the annealing in the vibration-free state. These two steps are one cycle.
Figure 4 shows the thermal cycle of the vibration PCR method.

Figure 4 – Thermal cycle of the vibration PCR method
In this study, we simply calculated assuming a single sphere of DNA. The vibrational energy
applied to DNA per vibrational cycle is expressed as the following equation.
𝜀𝑣,1𝑐𝑦𝑐𝑙𝑒 = 2𝑚𝜔2 𝐴2 = 8𝜋 2 𝑚𝐴2 𝑓 2(1)Here, m is total mass of DNA in the tube, A is vibrational amplitude of the tube, f is frequency ofthe tube. Considering the vibration time, it is expressed as the following equation.
𝜀𝑣 = 𝜀𝑣,1𝑐𝑦𝑐𝑙𝑒 𝑓∆𝑡 = 8𝜋 2 𝑚𝑓 3 𝐴2 ∆𝑡(2)In addition, since the DNA size used in this experiment is 500 bp, the polymer worm strand
model of the following equation considering anti-flexibility was applied rather than the complete
random coil model. For a DNA of q = 44 nm (full length L = 170 nm), the radius of inertia isestimated 35nm.
12𝑞3𝑞(3)√〈𝑆 2〉 = 𝐿𝑞 − 𝑞2 +[1 − (1 − 𝑒 −𝐿⁄𝑞 )]3𝐿𝐿
The number of DNA in the tube is on the order of 10 10 , and the maximum vibrational energy
applied per DNA sphere (including water) from the radius of inertia is estimated to be 9.1 × 10 −18 Jfrom equation (1).

3. EXPERIMENTAL METHODFirst, using a wave transducer, the entire microtube containing a solution of DNA (size 500 bp; bp= base pair) is vibrated. The vibration conditions at that time are a frequency of 100 to 800 Hz(amplitude of 0.44 to 0.015 mm), a voltage of 5 V, a vibration time of 10 seconds, and no vibration timeof 30 seconds, 5 cycles and 10 cycles.
Next, the size and amplification rate of DNA after vibration are measured by capillaryelectrophoresis.
Furthermore, the energy applied to the DNA in the tube from the outside is calculated from theanalysis results of capillary electrophoresis and the equations (1) to (3).

4. EXPERIMENTAL RESULT
Figure 5 shows the amplification rate at 5 cycles estimated by capillary electrophoresis, and Figure
6 shows the amplification rate at 10 cycles. Figure 7 shows the vibrational energy characteristic
calculated from equation (2).

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Amplification rate

2.00
1.80
1.60
1.40
1.20
1.00
0.80
0.60
0.400.20
0.00
100200300

400

500

600

700

800

700800Frequency［Hz］
Figure 5 – Amplification rate at 5 cycles
1.40

Amplification rate

1.20
1.00
0.80
0.60
0.40
0.20
0.00
100200

300

400
500
600
Frequency［Hz］

Figure 6 – Amplification rate at 10 cycles

Amplification rate

10 cycle

5 cycle

2.00
1.80
1.60
1.40
1.20
1.00
0.80
0.60
0.40
0.20
0.00

05101520

Vibrational energy 𝜀𝑣[×10-4J]
Figure 7 – Vibrational energy characteristics calculated from equation (2)48115. CONCLUSIONS
Amplification, rate was 1.8 times at the maximum. The vibration condition at that time was 800 Hz
for 5 cycles. The amplification rate was, as a whole, higher in 5 cycles than in 10 cycles. These results
indicate that when the vibrational energy applied to the DNA is too high, the enzyme is deactivated.

ACKNOWLEDGEMENTS
This research is funded by JSPS research grant JP18H03524.

REFERENCES
1. O. Kratky and G. Porod, Recl. Trav. Chim. Pays-Bas, 68, 1106 (1949).
2. E. S. Sobel and J. A. Harpst,Biopolymers, 31, 1559 (1991)

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