DNA storage device

Abstract

The present invention discloses a DNA storage device. In the first aspect of the present application, a storage device is provided, which includes: a storage strip, the storage strip including a strip base and a protective layer; a reaction tank, the reaction tank is used to accommodate a reaction liquid that reacts with the storage strip; and a read-write adjustment component, the read-write adjustment component is used to adjust the relative position of the storage strip and the reaction liquid so that the storage strip is immersed in the reaction liquid or separated from the reaction liquid. Through the arrangement of the reaction tank and the read-write adjustment component, when performing operations such as writing, reading, and erasing, it is only necessary to re-unroll the wound storage strip and put it into the read-write adjustment component to react, thereby avoiding permanent damage to the storage medium during operations such as reading while protecting the nucleic acid from damage by high temperature, free radicals, and water, and can be reused repeatedly.

Description

Technical Field

[0001] The present application relates to the field of data storage technology, and in particular to a DNA storage device. Background Art

[0002] With the rapid development of contemporary information technology, the amount of data and information is growing exponentially, a trend that will soon outstrip the capacity of existing storage media. Currently, the main storage methods used include magnetic, optical, and solid-state memory. These systems have increasingly exposed shortcomings, including short effective storage times, susceptibility to environmental influences, loss and corruption of data, high energy consumption of production equipment, and environmental pollution. In contrast, deoxyribonucleic acid (DNA), a natural information storage medium, has attracted widespread attention for its exceptionally secure storage capacity for the vast amount of genetic information from virtually every living organism in the world. As the densest and most stable data storage medium known, DNA boasts high storage density, long storage times, excellent parallel accessibility, and strong compatibility.

[0003] With the rapid development of DNA storage in recent years, various media and methods for DNA storage have been reported, including lyophilized powder, solid-phase substrates, silica spheres, and magnetic beads. While these media have demonstrated significant success in DNA storage, their limited functionality, the need to destroy the storage medium to access data, and the complex experimental procedures required for file indexing and decoding have limited their application in the DNA storage field. Sharon et al. attempted to load DNA dry powder onto a specific glass plate for long-term storage. They then used a digital microfluidics device to rehydrate the dry nucleic acid powder and extract it. While this method was able to extract the desired file, the extraction process resulted in a loss of dry powder, which in turn consumed the storage medium. Grass et al. used silica spheres containing nucleic acids to protect the nucleic acids from damage by heat, free radicals, and water. However, this method required hydrofluoric acid to etch the silica shell before extracting the nucleic acid file, permanently damaging the storage medium and rendering it unreusable. Furthermore, random access to the file was not possible.

[0004] Regarding file indexing, James et al. modified silica microspheres containing DNA files with primers with different sequences to label the files. Hybridization was performed using complementary chains with different fluorescent molecules. Fluorescence sorting was used to sort the microspheres with different fluorescent molecules to achieve random indexing of the files. While this method can achieve random access through the design of primers and fluorescent species, the primer modification process is complex, and fluorescence sorting requires the assistance of large-scale equipment. Most importantly, the silica microspheres cannot be recovered after the files are read, resulting in loss of the storage medium. Therefore, it is necessary to provide a storage medium that can be repeatedly written and erased and capable of random indexing. Summary of the Invention

[0005] The present application aims to solve at least one of the technical problems existing in the prior art. To this end, the present application proposes a storage medium that can be repeatedly written and erased multiple times and can perform random indexing.

[0006] In a first aspect of the present application, a storage device is provided, comprising:

[0007] A storage strip comprising a strip base and a protective layer; an index layer disposed on the strip base, the index layer comprising a plurality of index posts arranged to form an index pattern; the strip base between the index posts being used to immobilize oligonucleotides, which are used to specifically bind to the nucleic acid fragments to be stored; and a protective layer located on a side of the strip base away from the index layer and configured to enclose the index posts when the storage strip is wound.

[0008] A reaction tank, the reaction tank is used to accommodate a reaction liquid that reacts with the storage strip;

[0009] The read-write adjustment component is used to adjust the relative position of the storage strip and the reaction liquid, so that the storage strip is immersed in the reaction liquid or separated from the reaction liquid.

[0010] The storage device according to the embodiment of the present application has at least the following beneficial effects:

[0011] (1) The index pattern formed by the index column can divide the strip into several different areas. Different nucleic acid fragments can be combined with the oligonucleotide tags on the strip base in the index column in different partitions, thereby achieving the purpose of partition storage. Furthermore, the corresponding area can be identified according to the index pattern of different partitions to achieve the purpose of random indexing.

[0012] (2) Through the use of oligonucleotides, the nucleic acid fragments to be stored can be specifically bound to the storage strips for writing. Furthermore, under certain external conditions, such as a specific pH or enzyme, the nucleic acid fragments can be detached from the storage strips for reading or direct erasure. Since the oligonucleotides can still be retained, multiple lossless writes and erases can be performed, achieving the goal of WRMR (Write Multiple Read Multiple).

[0013] (3) By setting up the index column and the protective layer, the oligonucleotide and the nucleic acid fragment to be stored to be bound thereto are sealed in a winding manner, thereby improving the accuracy of storage.

[0014] (4) Through the setting of the reaction tank and the read-write adjustment component, when performing operations such as writing, reading, and erasing, it is only necessary to re-unroll the wound storage strip and put it into the read-write adjustment component to react. This avoids permanent damage to the storage medium during operations such as reading while protecting the nucleic acid from damage by high temperature, free radicals, and water, and can be reused many times.

[0015] In some embodiments of the present application, the strip substrate is a flexible substrate. In order to fix the oligonucleotide, the flexible substrate is preferably a flexible substrate capable of chemically bonding with nucleic acids, including but not limited to nylon membrane, modified nylon membrane (such as carboxyl modified), flexible gold membrane, flexible platinum membrane, etc.

[0016] In some embodiments of the present application, the index pillar is a photoresist pillar.

[0017] In some embodiments of the present application, the protective layer is a material that optionally has a protective effect when closing the index column, thereby improving the storage strip's resistance to oxidation, free radicals, water vapor, high and low temperature, UV, sunlight, enzyme reaction, shock, rubbing, bending and compression, etc., specifically including but not limited to organic protective layers, inorganic protective layers, organic-inorganic composite protective layers, etc., for example, a protective layer made of at least one of polyformaldehyde, polyether, polyacrylic acid, polyacrylamide, polypropylene, polyethylene, silica, calcium carbonate, calcium phosphate, metal organic framework, etc.

[0018] In some embodiments of the present application, the protective layer is an inorganic mineralized layer.

[0019] In some embodiments of the present application, the inorganic mineralized layer is a silica mineralized layer.

[0020] In some embodiments of the present application, the index pattern is selected from at least one of a barcode and a QR code.

[0021] In some embodiments of the present application, oligonucleotides are immobilized on the strip substrate between the plurality of index columns.

[0022] In some embodiments of the present application, the oligonucleotide is bound to the strip substrate via a chemical bond.

[0023] In some embodiments of the present application, the oligonucleotide may be a chemically modified nucleic acid molecule and / or an unmodified nucleic acid molecule. The unmodified nucleic acid molecule may be directly bonded to the strip substrate via a chemical bond, while the modified nucleic acid molecule may be bonded to the strip substrate via a chemical bond via a group or atom modified thereon.

[0024] In some embodiments of the present application, the chemical bonding method includes but is not limited to at least one of amino-carboxyl bonding, metal-sulfhydryl bonding, amino-aldehyde bonding, and coordination bonding between metal and nucleic acid.

[0025] In some embodiments of the present application, nucleic acid fragments are stored on the storage strip via fixed oligonucleotides.

[0026] Among them, the preparation method of the storage strip includes the following steps: taking a strip substrate, coating the surface of the strip substrate with photoresist, exposing and developing it through a mask with a preset index pattern to obtain an index layer having a plurality of photoresist columns, and the plurality of photoresist columns form an index pattern; and compounding a protective layer to the side of the strip substrate away from the index layer.

[0027] In some embodiments of the present application, nucleic acid fragments are stored on the storage strip of the storage device via fixed oligonucleotides.

[0028] In some embodiments of the present application, the read-write throttling component includes:

[0029] a first adjusting roller;

[0030] The second adjusting roller cooperates with the first adjusting roller to allow the storage strip to move above the reaction tank;

[0031] The control roller is located on the tape path of the storage strip and is configured to move in the up and down directions of the reaction tank so that the storage strip is immersed in the reaction liquid or separated from the reaction liquid.

[0032] In some embodiments of the present application, a reel-out roller and a reel-in roller are provided at both ends of the storage strip in the tape-traveling direction.

[0033] In some embodiments of the present application, the storage device further includes an identifier, which is used to identify an index pattern on the storage stripe.

[0034] A second aspect of the present application provides a storage method, the storage method comprising the following steps:

[0035] Providing a solution of nucleic acid fragments to be stored and the aforementioned storage device;

[0036] The solution of the nucleic acid fragment to be stored is injected into the reaction tank, and the position of the storage strip is adjusted by the read-write adjustment component, so that the storage strip is immersed in the solution to react, so that the nucleic acid fragment is specifically bound to the oligonucleotide of the storage strip.

[0037] In some embodiments of the present application, the solution further comprises dNTPs, a buffer component, a primer and a polymerase, and the reaction causes the nucleic acid fragment to specifically bind to the storage strip and form a complementary double-stranded nucleic acid fragment.

[0038] In some embodiments of the present application, the storage strip is further wound so that the protective layer encloses the index column.

[0039] In a third aspect of the present application, a reading method is provided, the reading method comprising the following steps:

[0040] providing a denaturing solution and a storage device storing nucleic acid fragments;

[0041] The denaturing liquid is injected into the reaction tank, and the storage strip is immersed in the denaturing liquid through the read-write adjustment component to react, so that the nucleic acid fragments are melted and released into the denaturing liquid. The denaturing liquid is recovered and sequenced.

[0042] In some embodiments of the present application, the nucleic acid fragment is a double-stranded nucleic acid fragment, including the nucleic acid fragment to be stored and the complementary fragment to the nucleic acid fragment to be stored. After the nucleic acid fragment is melted, the complementary fragment to the nucleic acid fragment to be stored is released into a denaturing solution. In some embodiments of the present application, the storage strip is indexed according to the index pattern to obtain a region of the storage strip with a preset index pattern. The region of the storage strip is immersed in the denaturing solution by a read / write adjustment component to react, causing the nucleic acid fragments in the region to melt and release into the denaturing solution. The denaturing solution is then recovered and sequenced.

[0043] In some embodiments of the present application, the sequencing method includes mixing the recovered denaturation solution with dNTPs, buffer components, primers and polymerase, recovering the free fragments in the denaturation solution and then sequencing.

[0044] In some embodiments of the present application, the step of decoding the base sequence of the sequencing result into a storage file for reading is also included.

[0045] A fourth aspect of the present application provides an erasure method, the erasure method comprising the following steps:

[0046] Providing a denaturing solution and the aforementioned storage device storing nucleic acid fragments;

[0047] The denaturing liquid is injected into the reaction tank, and the storage strip is immersed in the denaturing liquid for reaction through the read-write adjustment component. Then the denaturing liquid in the reaction tank is replaced with elution liquid and restriction endonuclease liquid in sequence, and the storage strip is eluted and enzyme-cut in sequence to remove the nucleic acid fragments on the storage strip, so that only oligonucleotides remain on the storage strip.

[0048] In some embodiments of the present application, the storage strip is indexed according to the index pattern to obtain the area of ​​the preset index pattern of the storage strip, and the area of ​​the storage strip is immersed in a denaturing liquid for reaction through the read-write adjustment component. Then, the denaturing liquid in the reaction tank is replaced with an elution liquid and a restriction endonuclease liquid in sequence, and the storage strip is eluted and enzyme-cut in sequence to remove the nucleic acid fragments on the storage strip, so that only oligonucleotides remain on the storage strip.

[0049] In response to the shortcomings of existing DNA storage media, such as single storage media functions, complex experimental operations required for file indexing and decoding, few storage partitions, the need to destroy the storage medium to read data, and the complex construction of DNA file indexing methods, which require the cooperation of large instruments, this application provides a nucleic acid storage strip and a storage device containing the storage strip, which can perform customized writing and lossless reading of DNA files. The special index pattern code design on the strip can quickly index the DNA files stored on the strip. In addition, the coiled layer structure formed by it has a huge storage capacity and can form a mineralized layer to protect DNA. Through the design of a micro-reaction tank, the entire process can be completed in a storage box similar to the size of a tape, without the need for large instruments.

[0050] Specifically, the storage strip has a two-layer structure. The upper layer is a nylon membrane substrate engraved with an index pattern, such as a barcode, and oligonucleotides, and the lower layer is a protective layer, such as silica. First, a series of index patterns are created on the upper strip substrate using photolithography. Files are partitioned according to the index patterns, and a high-speed barcode recognition camera can be used to index and locate the files. The silica protective layer on the lower strip forms a unique coiled structure. Once the storage strip is formed, the outward-curling lower silica layer fits tightly against the inward-curling upper nylon membrane layer, thereby protecting the DNA from environmental damage. Specific oligonucleotides are immobilized as primers on the activated nylon membrane layer, and a hybridization reaction allows DNA strands containing the file information to be written onto the nylon strip. Through DNA denaturation and polymerase extension reactions, the single-stranded DNA containing the file information is isolated for sequencing, readout, and lossless recovery. Most of the reactions in the writing and reading process occur within the reaction chamber within the device.

[0051] Additional aspects and advantages of the present application will be given in part in the description below, and in part will become obvious from the description below, or will be learned through practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 It is a structural diagram of a storage stripe in an embodiment of the present application.

[0053] Figure 2 It is a structural diagram of a storage device in an embodiment of the present application.

[0054] Figure 3 In the embodiment of this application Figure 2 A partial schematic diagram of the reaction tank of the storage device shown.

[0055] Figure 4 This is the DNA concentration test result of ten non-destructive readings of the storage device in Example 2 of the present application.

[0056] Figure 5 This is the test result of the data extraction accuracy of the storage device after four lossless reads in Example 2 of the present application.

[0057] Figure 6 This is the result of the influence of the DNA type on the storage strip on the accuracy of data extraction in Example 3 of the present application.

[0058] Figure 7 It is the barcode with two digits encoded on the storage strip in Example 3 of the present application.

[0059] Figure 8 This is the protective effect of the storage device on DNA in Example 4 of the present application.

[0060] Figure numerals: storage strip 100, protective layer 110, strip base 120, index layer 130, index column 131, oligonucleotide 132, shell 200, unwinding roller 210, winding roller 220, first adjustment roller 230, second adjustment roller 240, control roller 250, reaction tank 260, liquid inlet 261, liquid outlet 262, index pattern 270. DETAILED DESCRIPTION

[0061] The following will clearly and completely describe the concept and technical effects of this application in conjunction with the embodiments to fully understand the purpose, features and effects of this application. Obviously, the embodiments described are only part of the embodiments of this application, not all of them. Based on the embodiments of this application, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of this application.

[0062] The embodiments of the present application are described in detail below. The described embodiments are exemplary and are only used to explain the present application, and should not be understood as limiting the present application.

[0063] In the description of this application, "several" means more than one, "plurality" means more than two, "greater than," "less than," and "exceed" are understood to exclude the number itself, while "above," "below," and "within" are understood to include the number itself. The use of "first" and "second" in the description is solely for the purpose of distinguishing technical features and should not be construed as indicating or implying relative importance, implicitly specifying the number of the indicated technical features, or implicitly specifying the order of the indicated technical features.

[0064] In the description of this application, reference to the terms "one embodiment," "some embodiments," "illustrative embodiments," "examples," "specific examples," or "some examples" means that the specific features, structures, materials, or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of this application. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in any appropriate manner in any one or more embodiments or examples.

[0065] refer to Figure 1 , shows the structure of the storage strip in an embodiment of the present application. The storage strip 100 includes a strip base 120. An index layer 130 is provided on one side of the strip base 120. The index layer 130 includes a plurality of index columns 131. The plurality of index columns 131 are configured to form a macroscopic index pattern based on parameters such as their own shape and size, as well as the spacing and shape of the intervals between them. The strip base 120 in the intervals between the plurality of index columns 131 is used to fix oligonucleotides 132. These oligonucleotides 132 are used to specifically bind to the nucleic acid fragments to be stored. The storage strip 100 is provided with a protective layer 110 on the other side of the strip base 120 relative to the index layer 130. When the storage strip 100 is wound, the protective layer 110 is configured on the index layer 130 of the stacked adjacent rolls of the storage strip 100, thereby sealing the index columns 131 in the adjacent rolls, thereby protecting the nucleic acids from damage by high temperature, free radicals and water, and extending the service life and shelf life of the storage strip. In some specific embodiments, oligonucleotides are immobilized on the strip substrate between the index columns, and nucleic acid fragments are further stored on the storage strip via the immobilized oligonucleotides.

[0066] In some specific embodiments, the index column 131 of the storage strip 100 is a photoresist column, that is, it is made of photoresist. The photoresist can specifically be a positive photoresist or a negative photoresist, and the photoresist layer is patterned by exposure and development to obtain a plurality of photoresist columns. The process of preparing the index column from the photoresist material is simple and convenient, and is usually a hydrophobic material, which can form a hydrophobic barrier for the nucleic acid fragments fixed within the index column interval, further protecting the waterproof performance of the nucleic acid storage and improving the accuracy of subsequent reading. Since negative photoresist is better in terms of wet resistance and corrosion resistance, and the denaturing liquid used in the subsequent reading and erasing process is preferably an alkaline solution, the photoresist preferably uses a negative photoresist. Further, the negative photoresist preferably uses SU-8 photoresist. Compared to other negative photoresists, SU-8 photoresist overcomes the problem of insufficient depth ratio of common photoresists when using UV lithography. It has very low absorption in the near-ultraviolet range, and the exposure dose received across the entire photoresist is uniform, resulting in thick film patterns with vertical sidewalls and a high aspect ratio. It also exhibits superior mechanical properties, chemical corrosion resistance, and thermal stability. This allows for more durable and effective storage of nucleic acid fragments to be stored in the storage strip 100. In some preferred embodiments, the photoresist is applied to the strip substrate 120 by spin coating to form a photoresist layer, achieving a more uniform coating thickness. In some preferred embodiments, the spin coating speed can be 1000 to 3000 rpm (round per minute), and the specific speed can be adjusted based on requirements for the thickness of the index layer 130, among other factors. After spin coating, a stable index column and index pattern are obtained through pre-baking, mask exposure, rinsing, and hard baking. The composition of the developer, exposure, specific parameters of hard baking, and other optional steps such as post-baking and residual adhesive removal can be adjusted or selected according to actual needs.

[0067] In some specific embodiments, the protective layer 110 is optionally a material that can protect the nucleic acid fragments stored therein when the index column 131 is closed, and improve the storage strip's anti-oxidation, anti-free radical, anti-water vapor, high and low temperature resistance, anti-ultraviolet, anti-sunlight, anti-enzyme reaction, shock resistance, anti-rubbing, flexural and compressive properties, etc., specifically including but not limited to organic protective layers, inorganic protective layers, organic-inorganic composite protective layers, etc., such as organic materials such as polyformaldehyde, polyethyl ether, polyacrylic acid, polyacrylamide, polypropylene, polyethylene, inorganic materials such as silica, calcium carbonate, calcium phosphate, and organic-inorganic composite materials such as metal organic frameworks.

[0068] In some specific embodiments, protective layer 110 is an inorganic mineralized layer. This can more effectively protect the nucleic acid fragments stored therein, improving storage fidelity. Furthermore, the inorganic mineralized layer is a silica mineralized layer. Using silica as the material for protective layer 110 effectively encapsulates the nucleic acid fragments within index post 131 after winding, improving the stability of storage strip 100 in terms of UV resistance, moisture resistance, and high temperature resistance.

[0069] In some specific embodiments, the index pattern is selected from at least one of a barcode and a QR code. A QR code utilizes specific geometric shapes arranged in a two-dimensional pattern, using black and white patterns as graphic identifiers to express information. In this embodiment, the index pattern is formed by projecting the index columns 131 and the spaces between them perpendicularly to the storage strip 100 as black and white geometric shapes to form a QR code. Similarly, a barcode utilizes multiple black bars and spaces of varying widths arranged in a certain pattern as graphic identifiers to express information. In this embodiment, the index pattern is formed by projecting the index columns 131 and the spaces between them perpendicularly to the storage strip 100 as black bars and spaces. After the index pattern is formed, different index patterns, or different positions of the index patterns, on the storage strip 100 correspond to different storage strip partitions, and nucleic acid fragments containing different information are stored in different partitions.

[0070] In some specific embodiments, the strip substrate 120 is a flexible substrate. To immobilize oligonucleotides, the flexible substrate is preferably a flexible substrate capable of chemically bonding with nucleic acids, such as a nylon membrane, a modified nylon membrane (e.g., a carboxyl group), a flexible gold membrane, a flexible platinum membrane, or other flexible substrate materials that can covalently bond with nucleic acid fragments. In some preferred embodiments, the strip substrate 120 can be activated to obtain more binding sites, such as by activation with ethyldimethylaminopropylcarbodiimide (EDAC) or EDC / NHS, thereby allowing the binding of more oligonucleotides with the same surface area, and further allowing the storage of more nucleic acid fragments.

[0071] In some specific embodiments, the oligonucleotide is a linear polynucleotide fragment consisting of 2 to 200 nucleotide residues connected by phosphodiester bonds. Preferably, the number of nucleotide residues in the oligonucleotide is 2 to 160, 2 to 130, or 2 to 100. In some embodiments, the oligonucleotide is chemically bonded to the strip substrate 120. The oligonucleotide can be a chemically modified nucleic acid molecule and / or an unmodified nucleic acid molecule. Unmodified nucleic acid molecules can be directly chemically bonded to the strip substrate 120, while modified nucleic acid molecules can be chemically bonded to the strip substrate 120 via modified groups or atoms. Furthermore, chemical bonding methods include, but are not limited to, at least one of amino-carboxyl bonding, metal-sulfhydryl bonding, amino-aldehyde bonding, and coordination bonding between metal and nucleic acid. In some preferred embodiments, the oligonucleotide is immobilized by forming a covalent bond with the strip substrate 120 via amino, carboxyl, or other groups thereon. Preferably, the oligonucleotide solution is spotted onto the strip substrate 120 in the interval between the index columns. Under certain conditions, the strip substrate 120 reacts to cause covalent binding of the oligonucleotide. After the reaction is completed, the strip substrate 120 can be washed with a typical washing solution composed of SSC (Saline Sodium Citrate) or SSPE (Saline Sodium Phosphate EDTA) and SDS (Sodium Dodecyl Sulfate) or other buffer solutions well known in the art to remove unbound oligonucleotides.

[0072] An embodiment of the present application further provides a method for preparing a storage stripe, the method comprising the following steps:

[0073] Taking a strip substrate, coating a surface of the strip substrate with photoresist, exposing and developing the surface through a mask with a preset index pattern to obtain an index layer having a plurality of photoresist columns, wherein the plurality of photoresist columns form an index pattern;

[0074] Laminating a protective layer to the side of the strip base away from the index layer;

[0075] Oligonucleotides are immobilized on the strip substrate within the spaces between the photoresist pillars.

[0076] The embodiments of the present application also provide a storage method, which includes the following steps: contacting a solution of the nucleic acid fragment to be stored with the oligonucleotides on the aforementioned storage strip to react, so that the nucleic acid fragment is specifically bound to the strip substrate. The nucleic acid fragment is contacted with the oligonucleotides on the storage strip to hybridize the two, thereby writing the nucleic acid fragment to the storage strip for storage. In some specific embodiments, the nucleic acid fragment to be stored is a single-stranded nucleic acid, and the solution used in the storage process also includes dNTPs, buffer components, primers, polymerase and other components, so that the polymerase is used to initiate the replication of the single-stranded nucleic acid fragment, and the dNTPs form complementary fragments of the nucleic acid fragment to obtain a double-stranded nucleic acid fragment. The primers used in the solution include but are not limited to template chains for recovering nucleic acid fragments, and can also be used as primers in the subsequent reading process for PCR on the extracted free fragment files. In some specific embodiments, dNTPs include deoxyribonucleotides such as dATP, dGTP, dCTP, dTTP, and the buffer components and specific concentrations can be appropriately adjusted according to the actual replication or other processes. Generally, in order to enhance the effect of PCR, some additive components can be further added, specifically but not limited to Mg. 2+ , dimethyl sulfoxide (DMSO), glycerol, formamide, bovine serum albumin (BSA), (NH4)2SO4, polyethylene glycol (PEG), gelatin, non-ionic detergents (such as Tween 20, Triton X-100), betaine, tetramethylammonium chloride (TMAC), etc. In some specific embodiments, after writing is completed, the storage strip is further wound so that the protective layer seals the index column, thereby "encapsulating" the stored nucleic acid fragments. Through this step, the stability of the storage strip in terms of anti-oxidation, anti-free radicals, anti-water vapor, high and low temperature resistance, anti-ultraviolet, anti-sunlight, anti-DNA enzyme reaction, anti-vibration, anti-rubbing, anti-bending and anti-compression can be greatly improved, so that the nucleic acid fragments therein can be stored more stably and accurately.

[0077] The embodiments of the present application also provide a storage strip storing nucleic acid fragments obtained by the above-mentioned storage method. In some specific embodiments, the nucleic acid fragments stored on the storage strip are double-stranded nucleic acid fragments, including the original nucleic acid fragments to be stored and the fragments complementary thereto. In some specific embodiments, the purpose of storing the nucleic acid fragments is to store the nucleic acid fragments themselves, or to store the nucleic acid files represented by the nucleic acid fragments, for example, by encoding the original storage information into a nucleic acid sequence in a specific manner. The specific encoding and decoding conversion methods can at least refer to Grass, RN, et al, Angew. Chem. Int. Ed., 54: 2552-2555. or patent documents such as CN112582030A and CN104850760A.

[0078] An embodiment of the present application further provides a method for reading the storage strip storing nucleic acid fragments, the method comprising the following steps:

[0079] A denaturing solution and the aforementioned storage strip storing nucleic acid fragments are provided, the nucleic acid fragments are contacted with the denaturing solution to react, the nucleic acid fragments are melted and released into the denaturing solution, the denaturing solution is recovered, and sequencing is performed.

[0080] In some specific embodiments, the denaturing solution is an alkaline solution, including but not limited to NaOH, KOH, and other alkaline solutions. Nucleic acids can be denatured by treatment with acids, bases, or heat, but thermal denaturation requires low nucleic acid concentrations and low salt concentrations. Strong acids can degrade nucleic acids, leading to information loss. Therefore, it is preferred to use an alkaline solution for denaturation during reading to avoid nucleic acid degradation.

[0081] In some specific embodiments, the nucleic acid fragment is single-stranded or has at least one single-stranded structure. It is understood that during storage or subsequent reading, the nucleic acid fragment can be converted into a double-stranded structure by adding a solution to react.

[0082] In some specific embodiments, the nucleic acid fragment is a double-stranded nucleic acid fragment, including the nucleic acid fragment to be stored and the complementary fragment of the nucleic acid fragment to be stored. After the nucleic acid fragment is denatured, the complementary fragment of the nucleic acid fragment to be stored is released into the denaturing solution.

[0083] In some specific embodiments, the aforementioned storage strip storing nucleic acids is indexed according to an index pattern to obtain a region of the storage strip with a predetermined index pattern. Nucleic acid fragments within the region are then contacted with a denaturing solution to cause them to melt and release into the denaturing solution. The denaturing solution is then recovered and sequenced. By setting the index pattern, nucleic acid fragments within a specific region are selected for reading, thereby achieving random indexing and reading.

[0084] In some specific embodiments, the sequencing method includes mixing the recovered denaturing solution with dNTPs, buffer components, primers and polymerase, using the complementary fragments in the denaturing solution as templates to restore the double-stranded DNA and then perform sequencing.

[0085] In some specific implementations, after sequencing is completed, the base sequence in the sequencing result is re-decoded into the original storage file for reading.

[0086] An embodiment of the present application further provides a method for erasing the storage strip storing nucleic acid fragments, the method comprising the following steps:

[0087] A denaturing liquid and the aforementioned storage strip storing nucleic acids are provided. The nucleic acid fragments are contacted with the denaturing liquid for reaction. After deformation, they are eluted with an elution liquid. After elution, they are reacted with a restriction endonuclease to remove the nucleic acid fragments on the storage strip. The denaturing liquid in the erasing method is similar to the denaturing liquid in the reading method. The nucleic acid fragments are denatured by the denaturing liquid to release the nucleic acid fragments. After elution to remove the residual denaturing liquid, the nucleic acid fragments are further reacted with the restriction endonuclease to further remove the nucleic acid fragments to be stored on the storage strip, so that only the original oligonucleotides remain on the storage strip. Erasure is completed and rewriting is possible, achieving the purpose of multiple writing, multiple reading, and multiple erasing.

[0088] In some specific embodiments, the nucleic acid fragment is a double-stranded nucleic acid fragment, including the nucleic acid fragment to be stored and the complementary fragment of the nucleic acid fragment to be stored. After the nucleic acid fragment is denatured, the complementary fragment of the nucleic acid fragment to be stored is released into the denaturing liquid, and the nucleic acid fragment to be stored on the storage strip is further removed, so that only the original oligonucleotide remains on the storage strip.

[0089] In some specific embodiments, the aforementioned storage strip containing nucleic acid is indexed according to an index pattern to obtain a region of the storage strip with a predetermined index pattern. Double-stranded nucleic acid fragments within the region are then exposed to a denaturing solution for reaction, eluted, and then reacted with a restriction endonuclease to remove the nucleic acid fragments to be stored from the storage strip. By setting the index pattern, nucleic acid fragments within a specific partition are selected for erasure, thereby achieving the purpose of random indexing and erasure.

[0090] The embodiment of the present application further provides a storage device, which includes the aforementioned storage stripe. Figure 2 and Figure 3 In some specific embodiments, the storage device further includes a reaction tank 260, which is used to accommodate reaction liquid for reacting with the storage strip 100, including but not limited to a solution of nucleic acid fragments to be stored, a denaturing solution, a washing solution, a restriction endonuclease solution, etc. In some specific embodiments, the reaction tank 260 includes a liquid inlet 261 and a liquid outlet 262. The reaction liquid is discharged into the reaction tank 260 through the liquid inlet 261, and the reaction liquid in the reaction tank 260 is discharged through the liquid outlet 262. In some preferred embodiments, the reaction tank 260 has a structure that is narrow at the bottom and wide at the top. This structural design allows the reaction liquid to be quickly flushed into the reaction tank 260 and the height of the incoming liquid to be precisely controlled. Specific structures with a narrow bottom and wide top include but are not limited to inverted pyramids, inverted cones, inverted truncated cones, inverted truncated cones, hemispherical shapes, bowls, or further spherical / hemispherical shapes at the bottom or other locations, or irregular variations of these regular shapes.

[0091] In some specific embodiments, the storage device further includes a read-write adjustment component, which is used to adjust the relative position of the storage strip and the reaction liquid in the reaction tank so that the storage strip can be immersed in the reaction liquid or separated from the reaction liquid. Figure 2 In some specific embodiments, the read / write adjustment assembly includes a first adjustment roller 230, a second adjustment roller 240, and a control roller 250. The first adjustment roller 230 and the second adjustment roller 240 cooperate on either side of the reaction tank 260 to allow the storage strip 100 to travel above the reaction tank 260 at a set speed. The control roller 250 is located along the travel path of the storage strip 100 and is configured to move up and down along the reaction tank 260 to allow the storage strip 100 to enter or separate from the reaction solution. In some specific embodiments, a reel-off roller 210 and a reel-off roller 220 are provided at each end of the travel direction of the storage strip 100. The rotation of the reel-off roller 210 and the reel-off roller 220 unwinds the storage strip 100 from one end. After a specific area of ​​the storage strip 100 reaches above the reaction tank 260, the control roller 250 immerses it in the reaction solution for reaction, and then rewinds it from the other end. In some specific embodiments, the storage device further includes an identifier (not shown in the figure), which identifies the index pattern 270 on the storage strip 100, thereby controlling a specific area on the storage strip 100 to react with the reaction liquid to complete operations such as writing, reading, and erasing.

[0092] In some specific embodiments, the storage device further includes a housing 200, and the reaction tank 260, the storage strip 100, and the read / write adjustment assembly are all disposed within the housing 200. The housing 200 can be made of a polymer material with good mechanical properties, such as polycarbonate, to provide the storage device with good impact resistance, heat resistance, and flame retardancy.

[0093] The present application will be further described below through specific examples.

[0094] Example 1

[0095] This embodiment provides a storage stripe and a storage device including the storage stripe.

[0096] The specific process of preparing the storage stripe is as follows:

[0097] A nylon film is used as the strip base material, and a silicon dioxide film with a thickness of 10 to 100 nm is coated on one side of the nylon film by magnetron sputtering technology as a protective layer.

[0098] Then, the nylon film was cut into the size of a 5-inch silicon wafer and attached to the surface of the silicon wafer (the silicon dioxide film layer was in contact with the silicon wafer). 2-20 mL of SU-8 (black GmC2060) photoresist was spin-coated on the nylon film at a spin-coating speed of 1000-3000 rpm, and pre-baked at 65°C and 95°C for 5-20 minutes. Then, the silicon wafer with nylon and the mask with the designed barcode pattern were placed in the photolithography machine at 200-600 mJ / cm 2 The silicon wafer with the nylon film is exposed to light at an exposure intensity of 5 to 150 seconds, and then post-baked at 65°C and 95°C for 5 to 20 minutes in sequence. The silicon wafer is placed in a developer to wash away the uncured glue, and then baked at 150°C for 5 to 20 minutes to further cure the cured photoresist columns on the nylon film. At this time, the SU-8 photoresist is cured to form a plurality of photoresist columns configured as a barcode pattern.

[0099] The prepared nylon tape with a barcode pattern was spliced ​​into a 50-m long tape using ultrathin tape (3M 853), placed in 5-20% w / v ethyldimethylaminopropylcarbodiimide (EDAC), activated for 5-30 minutes, and then rinsed with deionized water. 100-1000 pmol of amino-containing primers were applied to the white area of ​​the barcode on the nylon membrane using a spotter and reacted at room temperature for 5-30 minutes. The unreacted primers were then washed away in 2×SSPE / 0.1% SDS at 40-60°C for 5-10 minutes to obtain a storage strip with oligonucleotides fixed on the nylon membrane within the gaps between the photoresist columns. The storage strip was then stored in 10-20 mM ethylenediaminetetraacetic acid (EDTA).

[0100] The storage device is further constructed by using the storage strip prepared above. Figure 2 The storage device includes a housing 200, which houses a payout roller 210 and a take-up roller 220. The storage strip 100 is wound into two rolls at both ends. One end is wound around the payout roller 210, then passes through a first adjustment roller 230, a control roller 250, and a second adjustment roller 240. The other end is then wound around the take-up roller 220, which is then rotated to rewind the strip. The payout roller 210 and the take-up roller 220 have diameters of 2 to 5 mm and are connected to a servo motor (not shown) to control the speed of the storage strip. The first and second adjustment rollers 230 and 240 control the direction of the storage strip's movement. The control roller 250, with a diameter of 0.5 to 2 mm, is located above the reaction tank 260. The control roller 250 allows the tiny document area on the storage strip 100 to come into contact with the reaction solution in the reaction tank 260.

[0101] The specific structure of the reaction tank 260 is as follows Figure 3As shown, the reaction tank has a chamfered design with a narrow bottom and wide top, allowing for rapid injection of liquid and precise control of the height of the liquid entering. The liquid inlet 261 and outlet 262 are used to input and output the reaction liquid from the reaction tank 260. Both the storage device and the micro-reactor are machined using CNC machine tools.

[0102] This embodiment also provides a method for multiple writing and erasing of nucleic acid files on a storage strip on the above storage device, specifically as follows:

[0103] A mixture of different encoded single-stranded DNA files, reaction buffer, dNTPs, primers, and DNA polymerase is introduced into the reaction tank at a certain concentration ratio, and the storage strip is immersed in the mixture through a control roller. After reacting at a temperature of 40-60°C for 30-60 seconds, the temperature is raised to 70-90°C and the reaction is continued for 30-60 seconds to fix the single-stranded DNA file on the storage strip and synthesize its complementary chain, completing the writing of the single-stranded DNA file.

[0104] When it is necessary to erase the written single-stranded DNA file, a 0.1-1M concentration of NaOH denaturing solution is passed into the reaction tank, and the storage strip is immersed in the denaturing solution through a control roller to denature the DNA. The denaturing solution in the reaction tank is then replaced with deionized water, and the denatured area of ​​the storage strip is eluted. After elution, the deionized water is replaced with a solution containing a DNA restriction endonuclease, thereby cutting off the DNA file area on the primer in the denatured area of ​​the storage strip, completing the data erasure on the storage strip.

[0105] Repeat the above writing and erasing process to perform multiple erasing and writing.

[0106] The following describes the indexing process of the storage device provided in this embodiment:

[0107] The storage device's unwinding roller is also connected to a servo, which, through an electrical connection to a barcode rapid identifier (PHOCUS-1801M), completes the indexing of large numbers of DNA files. The specific process is as follows: the servo connects to the unwinding roller, confirms the partition and location name to be indexed (i.e., the pre-index area), and simultaneously activates the servo and barcode rapid identifier to begin operation. The storage strip rotates at a speed of 2 to 20 cm / s. Once the identifier identifies the pre-index area through the barcode on the storage strip, the servo controls the unwinding roller to switch to a slow speed mode, adjusting the speed of the storage strip to 0.1 to 1 cm / s. At this point, the control roller is adjusted downward, placing the DNA files in the pre-index area on the storage strip into the reaction tank, completing the file indexing process. Writing, erasing, and reading operations can then be performed.

[0108] This embodiment also provides a method for losslessly reading a nucleic acid file on a storage strip on the above storage device, the method comprising the following steps:

[0109] After the DNA file is indexed, a 0.1-1M sodium hydroxide denaturing solution is introduced into the reaction tank for 5-30 minutes. The denaturing solution exiting the outflow channel is recovered and adjusted to neutrality, which is then stored as the sequencing solution. The sequencing solution is then placed into the sequencer for sequencing. Finally, the sequencing results are decoded and the file information is read, completing the entire non-destructive reading process.

[0110] After the denaturing liquid flows out of the reaction tank, a certain amount of reaction buffer, dNTPs, DNA polymerase and primers are reintroduced and reacted at 40-60°C for 5-15 minutes to restore the denatured single-stranded DNA to double-stranded nucleic acid fragments and preserve them again.

[0111] Example 2

[0112] Take the storage device in Example 1, wherein the storage strip is provided with eight partitions a1 to a8, and several photoresist columns in each partition constitute a complete barcode. There are several spacers between the photoresist columns in each barcode for fixedly storing DNA files. The spacers in each barcode are numbered from left to right on the path of the tape as the first spacer storage area, the second spacer storage area, the third spacer storage area, and so on. The image file is stored in 1000 to 8000 DNAs of 100 to 200bp, divided into 8 parts according to the writing method in Example 1, and written into the third spacer storage area of ​​the eight partitions a1 to a8 respectively. Wherein, the encoding and decoding methods refer to Yaniv Erlich, Dina Zielinski. DNA Fountain enables a robust and efficient storage architecture. Science, 03 Mar 2017, 355(6328): 950-954,.

[0113] After writing is completed, the data is extracted from the third interval storage area of ​​the barcode numbered a1 to a8. This is repeated four times. The content of each extracted DNA is measured using NanoDrop, and the sequence decoding and restoration file are sequenced. The detection results of the DNA content are as follows: Figure 4 As shown in the figure, it can be seen that the single-stranded DNA copy number is 10 8 ~10 10 The concentration required for sequencing is fully met. Figure 5 As shown in the figure, the image pixel loss rate is less than 2%.

[0114] Example 3

[0115] In order to obtain the maximum storage capacity of the DNA storage strip, the storage capacity of the DNA storage strip was calculated, and a file extraction accuracy greater than 90% was considered acceptable. First, the storage capacity of each white storage area within each barcode pattern of the storage strip finally prepared in Example 1 was calculated. Different numbers of 120bp DNA data files (one for each type) were written into each storage area, and the final extraction accuracy of different DNA types in the area was tested. The results are shown in Figure 2. Figure 6 As shown, each storage area can be inoculated with a maximum of 600 different types of 120bp DNA data files, which can be used to calculate a two-digit code (for example, letter + number code, a1, a2, b1, b2, c1, c2, etc., refer to Figure 7 The maximum storage capacity of a barcode (where the white and black areas in each barcode are approximately 50% of the total barcode area) is 703.125KB, while the storage capacity of a 50m DNA storage tape (rolled up to form a circle with a diameter of approximately 6cm) is 2.01GB.

[0116] Example 4

[0117] The storage device prepared in Example 1 was placed in an environment with a temperature of 65°C, a humidity of 75% and irradiation with fluorescent light. The data retention of the storage device was tested at different treatment times to observe the protection effect of the DNA storage device on DNA. A DNA solution under the same conditions was used as a control. The results are shown in FIG. Figure 8 As shown, the unprotected DNA solution was completely depleted within a week, while the DNA storage device still had a decoding data accuracy rate of over 60% after two weeks, indicating that the storage device's roll structure and silica protective layer have excellent protection effects on DNA files.

[0118] Example 5

[0119] The image "The Great Wave off Kanagawa" and the document "The Book of Changes" were encoded in a computer, and the electronic information bits were converted into nucleic acid sequence information. After being synthesized by a DNA synthesis instrument, they were chemically modified and fixed on a strip using the method in Example 1 and indexed during use. The file was then extracted, and the DNA sequence information was obtained and decoded through high-throughput sequencing, successfully obtaining the original image and document with a 100% accuracy rate.

[0120] Therefore, it can be seen from the above embodiments that the DNA storage strip protected by this application can store not only pictures and documents, but also any files composed of electronic bits, including but not limited to text, pictures, videos, music, software programs, computer systems, etc.

[0121] The present application has been described in detail above with reference to the embodiments. However, the present application is not limited to the above embodiments. Various modifications can be made within the scope of knowledge possessed by a person of ordinary skill in the art without departing from the purpose of the present application. In addition, the embodiments of the present application and the features of the embodiments can be combined with each other unless there is a conflict.

Claims

1. A storage device, characterized in that include: A storage strip comprises a strip base and a protective layer; the strip base is provided with an index layer, the index layer comprising a plurality of index posts, the plurality of index posts being configured to form an index pattern, the strip base between the plurality of index posts being used to immobilize oligonucleotides, the oligonucleotides being used to specifically bind to the nucleic acid fragments to be stored; the protective layer is located on a side of the strip base away from the index layer, and is configured to enclose the index posts when the storage strip is wound; The preparation method of the storage strip comprises the following steps: taking a strip substrate, coating a surface of the strip substrate with photoresist, exposing and developing the strip substrate through a mask with a preset index pattern to obtain an index layer having a plurality of photoresist columns, wherein the plurality of photoresist columns form an index pattern; a reaction tank, the reaction tank being used to accommodate a reaction liquid reacting with the storage strip; a read / write adjustment component, configured to adjust a relative position between the storage strip and the reaction liquid so that the storage strip is immersed in the reaction liquid or separated from the reaction liquid; The read-write regulation component includes: a first adjusting roller; a second adjusting roller, wherein the second adjusting roller cooperates with the first adjusting roller to enable the storage strip to move above the reaction tank; a control roller, the control roller being located on a tape path of the storage strip and being configured to move in an up-and-down direction along the reaction tank so as to immerse the storage strip in the reaction liquid or separate from the reaction liquid; A unwinding roller and a winding roller are respectively provided at both ends of the storage strip in the direction of tape travel. The storage strip is unwound from one end by the rotation of the unwinding roller and the winding roller. After a specific area of ​​the storage strip reaches the top of the reaction tank, the storage strip is immersed in the reaction liquid for reaction by the control roller, and is wound up from the other end. The unwinding roller and the winding roller are connected to a servo motor, so that the rotation speed of the storage strip can be controlled.

2. The storage device according to claim 1, wherein: The protective layer is any one of an organic protective layer, an inorganic protective layer, and an organic-inorganic composite protective layer.

3. The storage device according to claim 2, wherein: The material of the protective layer is selected from at least one of polyformaldehyde, polyether, polyacrylic acid, polyacrylamide, polypropylene, polyethylene, silicon dioxide, calcium carbonate, calcium phosphate, and metal organic framework.

4. The storage device according to claim 1, wherein Oligonucleotides are fixed on the strip substrate between the index columns. The storage device according to claim 1 , wherein: The storage device further includes an identifier configured to identify an index pattern on the storage stripe.

6. A storage method, characterized in that: The following steps are involved: Providing a solution of nucleic acid fragments to be stored and the storage device according to any one of claims 1 to 5; The solution of the nucleic acid fragment to be stored is injected into the reaction tank, and the position of the storage strip is adjusted by the read-write adjustment component so that the storage strip is immersed in the solution to react, and the nucleic acid fragment is specifically bound to the oligonucleotide of the storage strip.

7. The storage method according to claim 6, characterized in that: The method also includes winding the storage strip so that the protective layer encloses the index column.

8. A reading method, characterized in that The following steps are involved: Providing a denaturing solution and the storage device according to any one of claims 1 to 5 storing nucleic acid fragments; The denaturing liquid is injected into a reaction tank, and the storage strip is immersed in the denaturing liquid through a read-write adjustment component to react, so that the nucleic acid fragments are melted and released into the denaturing liquid, and the denaturing liquid is recovered and sequenced.

9. The reading method according to claim 8, characterized in that: The method also includes a step of decoding the base sequence of the sequencing result into a storage file for reading.

10. Erasing method, characterized in that, The following steps are involved: Providing a denaturing solution and the storage device according to any one of claims 1 to 5 storing nucleic acid fragments; The denaturing liquid is injected into the reaction tank, and the storage strip is immersed in the denaturing liquid for reaction through the read-write adjustment component. Then, the denaturing liquid in the reaction tank is replaced with an elution liquid and a restriction endonuclease liquid in sequence, and the storage strip is eluted and enzyme-cut in sequence to remove the nucleic acid fragments on the storage strip, so that only oligonucleotides remain on the storage strip.

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