High efficiency parallel DNA synthesis device using optical fiber bundle

Abstract

The present invention relates to an optical fiber-based DNA synthesis device capable of uniform light irradiation to an area where DNA is to be synthesized in a photochemical-based DNA synthesis process, and parallel DNA synthesis, wherein optical fiber strands are used as a light homogenizer and a DNA synthesis substrate.

Description

High-efficiency parallel DNA synthesis device using optical fiber bundles

[0001] The present invention relates to an optical fiber-based DNA synthesis device capable of evenly irradiating light to an area where DNA is to be synthesized in a photochemical-based DNA synthesis process and capable of parallel DNA synthesis.

[0002] The growth of personal social networking services (SNS), which primarily focus on images and videos, is driving a rapid increase in data generation. Data volume is expected to increase further as augmented reality (AR), virtual reality (VR), mixed reality (MR), and 3D media such as holograms become widespread. Specifically, the amount of data stored worldwide by 2040 is projected to reach 10 24 10 in bits 29 It is expected to reach bits (Summary report, Technology Working Group Meeting on future DNA synthesis technologies (September 14, 2017, Arlington, VA)).

[0003] Currently, the data storage medium of major devices such as computers and smartphones is flash memory, which has a data density of about 1 bit per 1 pg. In order to supply flash memory corresponding to the amount of data required in 2040, 10 14 kg of silicon wafers are needed, but the supply of silicon wafers in 2040 is expected to be 10 8 It is estimated to be around kg, which falls far short of demand. Furthermore, existing data storage media have a limited data retention period of around 10 years.

[0004] Accordingly, efforts are continuously being made to develop new data storage media, one of which is DNA storage devices. Using DNA as a storage medium can overcome the data storage density limitations of existing storage media and can store information reliably even under physical shock. Furthermore, 1 kg of DNA can store 10 times the amount of flash memory. 9 2x10 equivalent to kg 24 DNA storage devices are suitable for storing large amounts of data because they can store bits, and they offer the advantage of a very long data retention period. However, errors occurring during DNA synthesis can lead to reduced data storage density and information loss, hindering the commercialization of DNA storage devices.

[0005] In particular, photochemical DNA synthesis methods deprotect DNA terminal protecting groups by illuminating them with light. This causes light to reach undesired regions due to diffraction, scattering, and reflection. Furthermore, there are areas where light insufficiently reaches the desired DNA site. These problems result in deprotection occurring at unintended sites and failure to deprotect DNA at targeted sites, resulting in DNA synthesis errors.

[0006] In the above situation, the inventor of the present invention studied a method for evenly irradiating light to an area where DNA is to be synthesized in a photochemical-based DNA synthesis process, and devised a method for using an optical fiber bundle as a light homogenizer and a DNA synthesis substrate.

[0007] Accordingly, an object of the present invention is to provide a DNA synthesis device capable of implementing the above method and a DNA synthesis method using the device.

[0008] In order to achieve the above purpose, one aspect of the present invention is

[0009] light source;

[0010] A light homogenizer comprising a bundle of optical fibers, one end of which functions as a light input portion and the other end as a light output portion; and

[0011] A light-space modulator that controls the pattern in which a light source is irradiated to the input portion of the light homogenizer;

[0012] The output section of the above light homogenizer provides a DNA synthesis device that functions as a DNA synthesis substrate.

[0013] The above light source may be a micro LED (micro light emitting diode) configured to irradiate light onto a DNA synthesis substrate when synthesizing DNA.

[0014] In the present invention, the optical fiber may have a diameter of 0.1 μm to 1 mm.

[0015] Additionally, the light homogenizer made of a bundle of optical fibers is characterized by being freely bendable.

[0016] In the present invention, the light-space modulator is configured to control the pattern of light irradiated on a DNA synthesis substrate when synthesizing DNA, and a photomask, a digital micromirror device (DMD), or a micro light emitting diode (micro LED) may be used.

[0017] In the present invention, when microLEDs are used as a light source and a light-space modulator, the light source and the light-space modulator can be integrated within a DNA synthesis device.

[0018] Another object of the present invention is to provide a DNA synthesis method using the above DNA synthesis device, comprising the following steps:

[0019] (a) a step of irradiating light onto the input of a light homogenizer;

[0020] (b) a step of contacting the output of the light homogenizer with a DNA deprotection solution; and

[0021] (c) A step of bringing the output of the light homogenizer into contact with the coupling solution.

[0022] The method may further comprise the step of (d) contacting the output of the light homogenizer with an oxidizing solution.

[0023] The above step (b) may be a step of (b-1) moving the output portion of the light homogenizer to contact it with the DNA deprotection solution; or a step of (b-2) contacting the DNA deprotection solution while keeping the output portion of the light homogenizer fixed.

[0024] The above step (c) may be a step of (c-1) moving the output portion of the light homogenizer to contact it with the coupling solution; or a step of (c-2) contacting the coupling solution while keeping the output portion of the light homogenizer fixed.

[0025] Using a DNA synthesis device according to an example of the present invention, uniform light can be irradiated to a DNA synthesis area, thereby reducing DNA synthesis errors and also reducing the sample consumed for DNA synthesis.

[0026] Figure 1 schematically illustrates the causes of synthetic errors in the photochemical-based DNA synthesis process.

[0027] Figure 2 shows the results of measuring the amount of light irradiated to the synthesis site during a photochemical-based DNA synthesis process.

[0028] Figure 3 shows an example of the process in which DNA ends are deprotected and new nucleotides are added in a photochemical-based DNA synthesis method.

[0029] Figure 4 shows an example of a process in which DNA ends are deprotected and new nucleotides are added by a deprotecting molecule providing substance in a photochemical-based DNA synthesis process.

[0030] Figure 5 shows that when patterning is performed on a substrate covered with photoresist in a CMOS semiconductor process using a DMD (digital micromirror device), a blind spot exists in the post portion of the mirror in the DMD structure.

[0031] Figure 6 shows an example of DNA synthesis using an optical fiber bundle as a DNA synthesis substrate, irradiating uniform light and reducing sample consumption.

[0032] Figure 7 shows an example of fixing an optical fiber bundle and synthesizing DNA using microcontact printing.

[0033] Figure 8 shows an example of uniform light irradiation in a photochemical-based DNA synthesis process using a UV LED array and an optical fiber bundle.

[0034] Figure 9 shows an example of irradiating uniform light onto a DNA synthesis substrate in a photochemical-based DNA synthesis process using a waveguide plate corresponding 1:1 to each UV LED element of a UV LED array.

[0035] Hereinafter, with reference to the attached drawings, preferred embodiments will be described in detail so that those skilled in the art can easily practice the present invention. However, when describing preferred embodiments of the present invention in detail, if it is determined that a specific description of a related known function or configuration may unnecessarily obscure the gist of the present invention, the detailed description thereof will be omitted. In addition, the same reference numerals are used throughout the drawings for parts that have similar functions and actions. Meanwhile, "including" a certain component does not exclude other components unless specifically stated otherwise, but rather means that other components may be further included.

[0036]

[0037] Figure 1 schematically illustrates the causes of synthetic errors in the photochemical-based DNA synthesis process.

[0038] When light (UV or 100 to 500 nm wavelength) is selectively irradiated to the synthesis area of ​​the DNA synthesis substrate using a photomask or a digital micromirror device (DMD), the light may be reflected, scattered, or spread by diffraction from the DNA synthesis substrate, thereby reaching unnecessary areas.

[0039] As shown in Figure 1, light diffracted from the DNA synthesis substrate deprotects DNA located outside the DNA synthesis region. Furthermore, light insufficiently reaches the edges of the synthesis region in the DNA synthesis substrate, where stochastic deprotection of protecting groups can occur.

[0040] The area where light insufficiently reaches is indicated as an error prone zone in Figure 1. Since the light reaching the error prone zone does not reach the threshold required for deprotection of the protecting group, some protecting groups are deprotected, but some protecting groups remain as is without being removed.

[0041] It can also be confirmed through Figure 2 that there is an area where light insufficiently reaches the target location of the DNA synthesis substrate.

[0042]

[0043] Figure 2 shows the results of measuring the amount of light irradiated to the target location (DNA synthesis area) of the DNA synthesis substrate during a photochemical-based DNA synthesis process.

[0044] In Fig. 2, the left picture is a top view of light irradiating the DNA synthesis site, and the intensity of light at each point is expressed in color. The right picture is a drawing of the light intensity along the x-axis (blue) and y-axis (green). When light is shined on the DNA synthesis site, the central area maintains a constant amount of light, but the amount of light reaching the edge decreases with a gradient. In other words, the contrast ratio of the light pattern illuminated on the substrate is not perfect.

[0045] As described in the description of Fig. 1, the edge portion (corresponding to the error-prone area in Fig. 1) cannot provide sufficient light for deprotection because the light intensity gradually decreases, and thus deprotection may occur at certain locations but not at other locations. Since this probabilistic deprotection is very random, in each cycle of DNA synthesis, even if the DNA strand was not deprotected at the (n-1)th DNA synthesis, deprotection may occur at the nth DNA synthesis, and when a new nucleotide is added (coupled), a deletion error may occur for the (n-1)th sequence.

[0046] That is, whether or not deprotection occurs in the nth DNA synthesis is independent of whether or not deprotection occurred in the previous (n-1)th DNA synthesis. Because of deletion errors in these edges (error-prone regions), capping is required to block the ends of the DNA or nucleic acids being synthesized to prevent additional synthesis in each synthesis cycle, and a process is required to remove DNA shorter than the target length after synthesis is complete. The influence of these errors in the edges increases as the length to surface ratio increases, that is, as the size of the area irradiated with light decreases, which makes it difficult to reduce the size of a single spot to increase the number of possible simultaneous synthesis sites in microarray DNA synthesis.

[0047]

[0048] Figures 3 and 4 illustrate the deprotection and coupling mechanisms during DNA synthesis.

[0049] Figure 3 shows an example of the process in which DNA ends are deprotected and new nucleotides are added in a photochemical-based DNA synthesis method.

[0050] When the deprotecting group at the end of DNA is removed by light, an -OH group is exposed at the 5' end of the DNA, allowing new nucleotides to be incorporated into the end of the DNA strand being synthesized. Since the nucleotides supplied for synthesis are attached to a protecting group at one end, additional DNA synthesis will not occur without a separate deprotection process.

[0051] Protecting groups that fall off by direct reaction with light include BzNPPOC, NPPOC, and SPh-NOOPC.

[0052] Figure 4 shows an example of the process in which DNA ends are deprotected and new nucleotides are added by a deprotecting molecule supplier during a photochemical-based DNA synthesis process.

[0053] When exposed to light, the deprotecting molecule donor molecule reacts with light to release an active deprotecting molecule, which attacks and removes the protecting group. This exposes the -OH group at the 5' end of the DNA, allowing a new nucleotide to bind to the end of the DNA strand being synthesized.

[0054] The above deprotecting molecule providing substance is hydroquinone, and the active deprotecting molecule is hydrogen ion (H + ) can be used, and DMT can be used as a protector.

[0055]

[0056] Figure 5 shows that when patterning is performed on a substrate covered with photoresist in a CMOS semiconductor process using a DMD (digital micromirror device), a blind spot exists in the post portion of the mirror in the DMD structure.

[0057] In addition to the problem of insufficient light reaching the edge area described in Figure 1, the DMD's structural blind spot at the post area in the center of the mirror makes it impossible to achieve uniform UV exposure. That is, when light is patterned onto the surface of a DNA synthesis substrate using a DMD, the light intensity in the center area where the post is located is relatively low compared to the periphery (see intensity profile on the right). Consequently, sufficient light intensity required for DNA deprotection is not guaranteed, which can lead to deletion errors.

[0058]

[0059] FIG. 6 shows an example of a DNA synthesis device according to an embodiment of the present invention, specifically, a method of synthesizing DNA by irradiating uniform light and reducing sample consumption using an optical fiber bundle as a DNA synthesis substrate.

[0060] In FIG. 6, “UV source” corresponds to a light source in the DNA synthesis device of the present invention, “DMD” corresponds to a light-space modulator, and “optic fiber bundle” corresponds to a light homogenizer.

[0061] The above "light homogenizer" refers to a configuration that uniformly transmits light irradiated from a light-space modulator to a DNA synthesis substrate, and is composed of an optical fiber bundle as illustrated in FIG. 6. One end of the optical fiber bundle is a light input portion, and the other end is a light output portion. As can be seen in the inset in FIG. 6, even if light is not uniformly input to the light input portion, light is uniformly output from the light output portion because the light is totally reflected within the optical fiber. Therefore, in the light homogenizer, the number of light input portions and light output portions can be as many as the number of optical fibers. Through this configuration, the deletion error (FIG. 5) at the edge of the DMD pixel, which was a limitation of maskless array synthesis using DMD, can be suppressed.

[0062] Meanwhile, the output portion of the above-mentioned light homogenizer (optical fiber) also functions as a substrate for DNA synthesis. Therefore, the end of the optical fiber can be used as the output portion, or the end of the optical fiber can be coated with a material that facilitates DNA attachment and used as the output portion.

[0063] The DNA synthesis device of the present invention can control the light pattern irradiated to each optical fiber bundle by a light-space modulator, so that DNA can be synthesized in parallel as many as the number of strands of optical fiber.

[0064] In the present invention, the optical fiber may have a diameter of 0.1 μm to 1 mm, and the length of the optical fiber strand may be 0.1 μm to 1 m. In addition, the number of optical fiber strands may be 10 2 10 inland 10 It could be a range.

[0065] The above optical fiber may be made of glass, but any material that can be made into an optical fiber may be used without limitation.

[0066] Referring to FIG. 6, the DNA synthesis process using the DNA synthesis device of the present invention comprises the following steps:

[0067] (a) a step of irradiating light onto the input of a light homogenizer;

[0068] (b) a step of contacting the output of the light homogenizer with a DNA deprotection solution; and

[0069] (c) A step of bringing the output of the light homogenizer into contact with the coupling solution.

[0070] The method may further comprise the step of (d) contacting the output of the light homogenizer with an oxidizing solution.

[0071] More specifically, the step of irradiating light to the input portion of the light homogenizer (a) can be performed after irradiating light from a light source to a light-space modulator, and light can be irradiated only to a specific optical fiber among the optical fiber bundles forming the light homogenizer through the light-space modulator.

[0072] The above step (b) is a step of deprotecting the protecting groups such as DMT and BzNPPOC at the end of DNA so that new monomers can be combined.

[0073] (b-1) a step of moving the output of the light homogenizer to contact it with the DNA deprotection solution; or

[0074] (b-2) A step of contacting the DNA deprotection solution with the output section of the light homogenizer in a fixed state;

[0075] The above DNA deprotection solution may be a solution commonly used in the field of DNA synthesis technology.

[0076] In addition, the above step (c) is a step of binding a new monomer to the deprotected DNA end,

[0077] (c-1) a step of moving the output portion of the light homogenizer to contact the coupling solution; or

[0078] (c-2) A step of contacting the coupling solution with the output section of the light homogenizer while it is fixed;

[0079] The above DNA coupling solution may be a dA, dG, dC, or dT solution commonly used in the field of DNA synthesis technology.

[0080] As can be seen from the description of steps (b) and (c) above, when performing the deprotection step or the coupling step, it is also possible to configure the optical fiber bundle in a bent shape rather than a straight shape. By configuring it in such a bent shape, when irradiating light to the input portion of the optical fiber bundle, the light irradiated to the cladding portion rather than the core portion can be suppressed from reaching the output portion. For example, the connection portion from the input portion of the optical fiber bundle to the output portion can be bent by more than 30 degrees.

[0081] Using the DNA synthesis device of the present invention, DNA can be synthesized in the same manner as in the conventional method by bringing the output portion of the optical fiber bundle into contact with the synthesis surface of a flow cell. However, DNA can also be synthesized by sequentially bringing the output portion of the flexible optical fiber bundle into contact with a bulk synthesis solution by moving it (red dotted arrow in Fig. 6) without constructing a flow cell. By moving the output portion of the optical fiber bundle in this way and synthesizing DNA on the surface of the output portion, the solution can be used repeatedly, thereby minimizing solution consumption.

[0082] For example, using a DMD, a specific optical fiber is selectively illuminated, and then the optical fiber bundle is moved to contact a coupling solution, thereby coupling monomers. This cycle of moving the optical fiber bundle again to contact an oxidizing solution to stabilize the DNA terminal structure is repeated, allowing DNA synthesis.

[0083] Meanwhile, the DNA synthesis method of the present invention may include a step of washing the output unit each time steps (a) to (d) are repeated.

[0084]

[0085] Figure 7 shows an example of fixing an optical fiber bundle and synthesizing DNA using microcontact printing.

[0086] Synthesizing DNA according to Figure 7 can reduce the consumption of each solution required for synthesis, and the output portion of the optical fiber is represented as a DNA synthesis substrate. In microcontact printing, materials such as PDMS (poly dimethyl siloxane) and hydrogel can be used as microstamp materials.

[0087] This method can suppress deletion errors compared to optical-based synthesis using existing flow cells, reduce the cost required for DNA synthesis, and minimize environmental pollution by reducing the use of organic solvents.

[0088]

[0089] Figure 8 shows an example of uniform light (UV) irradiation in a photochemical-based DNA synthesis process using a UV LED array and an optical fiber bundle.

[0090] UV LEDs used as light sources have the advantage of being easy to control each LED through a circuit when implemented in the form of microLEDs, and of lower cost compared to using UV lasers or UV HE lamps.

[0091] However, since each UV LED element is large compared to the size of the DMD pixel, they can be integrated on a micro scale using optical fiber bundles. That is, in Fig. 7, the light source and the light-space modulator are integrated into a micro LED, and the input of each optical fiber bundle is connected to each UV LED element, and the output is connected to a flow cell, enabling DNA synthesis.

[0092] Even if the UV LED does not correspond exactly to the light input of the optical fiber bundle, the optical fiber acts as a light pipe, i.e., a homogenizer (lower right figure in Fig. 7), so that a constant light is output from the light output, enabling high-efficiency parallel DNA synthesis.

[0093]

[0094] Figure 9 shows an example of irradiating uniform light onto a DNA synthesis substrate in a photochemical-based DNA synthesis process using a waveguide plate corresponding 1:1 to each UV LED element of a UV LED array.

[0095] Fig. 8 shows that optical fibers are connected to UV LEDs one by one to irradiate light evenly, but Fig. 9 shows that a waveguide plate is used so that when placed directly on a UV LED array, the part corresponding to the optical fiber can be positioned directly on each UV LED element without separate alignment.

[0096] In Fig. 9, the UV LED and the waveguide plate are depicted with a gap between them, but in actual applications, the UV LED and the waveguide plate are used in close contact.

Claims

1. Light source; A light homogenizer comprising a bundle of optical fibers, one end of which functions as a light input portion and the other end as a light output portion; and A light-space modulator that controls the pattern in which a light source is irradiated to the input portion of the light homogenizer; A DNA synthesis device in which the output portion of the above light homogenizer functions as a DNA synthesis substrate.

2. A DNA synthesis device according to claim 1, wherein the optical fiber has a diameter of 0.1 μm to 1 mm.

3. A DNA synthesis device according to claim 1, wherein the light homogenizer is bendable.

4. A DNA synthesis device according to claim 1, wherein the light-space modulator is a photomask, a digital micromirror device (DMD), or a micro light emitting diode (micro LED).

5. A DNA synthesis device according to claim 1, wherein the DNA synthesis device is a DNA synthesis device in which a light source and a light-space modulator are integrated into a microLED.

6. A DNA synthesis method using the DNA synthesis device of paragraph 1, comprising the following steps: (a) a step of irradiating light onto the input of a light homogenizer; (b) a step of contacting the output of the light homogenizer with a DNA deprotection solution; and (c) A step of bringing the output of the light homogenizer into contact with the coupling solution.

7. A DNA synthesis method according to claim 6, wherein the method further comprises the step of (d) contacting the output of the light homogenizer with an oxidizing solution.

8. In paragraph 6, step (b) (b-1) a step of moving the output of the light homogenizer to contact it with the DNA deprotection solution; or (b-2) A step of contacting a DNA deprotection solution with the output section of a light homogenizer while it is fixed; a DNA synthesis method.

9. In paragraph 6, step (c) (c-1) a step of moving the output portion of the light homogenizer to contact the coupling solution; or (c-2) A step of contacting a coupling solution with the output of a light homogenizer while it is fixed; a DNA synthesis method.

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