An automated in situ DNA information storage and reading method

Abstract

The application discloses an automatic in-situ DNA information storage and reading method, which comprises the following steps: (1) converting a data file to be stored into a ternary number through information exchange code; (2) encoding the ternary number into a corresponding DNA sequence according to a correspondence coding table of the ternary number and DNA base conversion; (3) synthesizing the corresponding DNA sequence encoded in step (2) on a digital microfluidic chip by using a DNA synthesis method to store information of the data file; (4) sequencing the DNA sequence synthesized in step (3) on the digital microfluidic chip by using a DNA sequencing method to read the information of the data file; and (5) decoding and converting the information read in step (4) according to the correspondence coding table. The application can provide an integrated solution for writing and reading of DNA information storage.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of information storage and reading, and particularly relates to an automatic in-situ DNA information storage and reading method. BACKGROUND

[0002] Currently, the Internet and information technology are in a rapid changing and flourishing development stage, and human society is experiencing an unprecedented information explosion era. It is estimated that by 2025, the amount of data generated globally per day will reach 463EB (1EB≈106TB), and will further increase. The gap between this and the capacity of traditional data storage media is expanding exponentially. Traditional information storage resources will be exhausted in the future. Therefore, seeking new information storage media and modes has become a problem to be solved.

[0003] Compared with traditional optical or magnetic storage media, DNA has ultra-high storage density (1g of DNA can store 215 million GB; a bacterium can store 10 3 TB of capacity), ultra-long data stability (the half-life of DNA can exceed 500 years; DNA stored in the doomsday seed vault can exceed 2 million years; DNA as a preservation medium does not have the problem of technical obsolescence or incompatibility), and theoretically ultra-high read-write speed (<100μs / bit), ultra-low energy consumption (<10 -10 W / GB), making it a potential information storage medium for the next generation.

[0004] In the past few decades, the development and progress of DNA single-base resolution read-write technology (DNA synthesis and sequencing technology) and digital information coding theory have laid a solid foundation for DNA-based information data storage strategies. The basic concept of DNA data storage can be traced back to the mid-1960s, after J. Watson and F. Crick revealed the structure of DNA as a carrier of genetic information, many researchers proposed the concept of storing specific information in DNA. Limited by the still nascent DNA synthesis technology and DNA sequencing technology at that time, these early works were not practical. Until 2012-2013, G. Church et al. (SCIENCE, 2012, 337(6102): 1628) and N. Goldman et al. (NATURE, 2013, 494: 77-80) used more mature DNA synthesis (high-throughput chemical synthesis technology) and sequencing technology (second-generation sequencing technology) to make DNA information storage a milestone in practical application. Since then, researchers have gradually completed the whole process of DNA information storage strategy, including encoding, synthesis, storage, retrieval, sequencing, and decoding. There have been major breakthroughs and progress in each link (NAT. REV.. GENET., 2019, 20: 456-466).

[0005] However, DNA synthesis-based information storage also faces great challenges. First, the current DNA information storage based on DNA chemical synthesis method and second-generation sequencing technology has a high error rate in reading and writing. The average error rate of existing DNA chemical synthesis method is about 0.7% (NAT. COMMUN., 2020, 11: 5345). However, as the length of DNA synthesis increases, the accuracy will decrease significantly. For example, the accuracy of adding one nucleotide is about 99.5%. Therefore, the final accuracy of a 200-base DNA chain is only 36.7% (NUCLEIC ACIDS RES., 2021, 49, 10: 5451). More errors come from DNA sequencing. The current error rate of second-generation sequencing is about 1%-2.5%, and the error rate of third-generation sequencing is higher than 10% (NAT. BIOTECHNOL., 2018, 36: 242-248). Second, most of the current methods rely on complex and large DNA synthesis, amplification, and sequencing instruments, which are difficult to operate and require professional personnel to operate, further increasing the cost and error rate of DNA information storage (SCI. ADV., 2021, 7(46): eabk0100). Finally, there is a serious lack of integrated platforms and instruments specifically designed for DNA information storage. The stages of DNA information storage are completed by different devices, and encoding and decoding cannot be performed in situ. Moreover, it is difficult to achieve automation, which cannot meet the needs of high efficiency and low cost industrialization. SUMMARY

[0006] The present application aims to overcome the defects of the prior art and provide an automatic in-situ DNA information storage and reading method.

[0007] The technical solution of the present application is as follows:

[0008] An automatic in-situ DNA information storage and reading method, comprising the following steps:

[0009] (1) converting a data file to be stored into ternary digits through information exchange codes;

[0010] (2) encoding the ternary digits into corresponding DNA sequences according to a correspondence coding table between the ternary digits and DNA base transformations;

[0011] (3) synthesizing the corresponding DNA sequences encoded in step (2) on a digital microfluidic chip by using a biological enzyme-catalyzed DNA synthesis method, a chemical phosphoramidite DNA synthesis method or an enzyme ligation DNA synthesis method to store the information of the data file;

[0012] (4) sequencing the DNA sequences synthesized in step (3) on the digital microfluidic chip by using a DNA sequencing method to read the information of the data file;

[0013] (5) decoding and converting the information read in step (4) according to the correspondence coding table.

[0014] In a preferred embodiment of the present application, the information exchange codes include words, pictures, music and videos.

[0015] Further preferably, the words include ASCII and GBK, the pictures include JPG and BMP, the music includes MAV and MP3, and the videos include MPEG and H.26X.

[0016] In a preferred embodiment of the present application, the correspondence coding table is implemented by coding and decoding 0, 1 and 2 of ternary digits according to transformations between A, T, C and G, four bases in DNA sequences, wherein,

[0017] A→C, T→G, C→A and G→T are all converted into 0;

[0018] A→T, T→C, C→G and G→A are all converted into 1;

[0019] A→G, T→A, C→T and G→C are all converted into 2;

[0020] A→C represents a case where the next position of A in a DNA sequence is C,

[0021] T→G represents a case where the next position of T in a DNA sequence is G,

[0022] C→A represents a case where the next position of C in a DNA sequence is A,

[0023] G→T represents a case where the next position of G in a DNA sequence is T,

[0024] A→T represents a case where the next position of A in a DNA sequence is T,

[0025] T→C represents a case where the next position of T in a DNA sequence is C,

[0026] C→G represents a case where the next position of C in a DNA sequence is G,

[0027] G→A represents a case where the next position of G in a DNA sequence is A,

[0028] A→G represents a case where the next position of A in a DNA sequence is G,

[0029] T→A represents a case where the next position of T in a DNA sequence is A,

[0030] C→T represents a case where the next position of C in a DNA sequence is T,

[0031] G→C represents a case where the next position of G in a DNA sequence is C.

[0032] In a preferred embodiment of the present application, the biological enzymatic DNA synthesis method is a terminal transferase-based enzymatic DNA synthesis method, a polynucleotide phosphorylase-based DNA chemical synthesis method, or a T4 RNA ligase-based DNA synthesis method.

[0033] In a preferred embodiment of the present application, the DNA sequencing method is a pyrophosphate DNA sequencing method, a Sanger sequencing method, a second-generation sequencing method, or a nanopore third-generation sequencing method.

[0034] In a preferred embodiment of the present application, the digital microfluidic chip has an upper plate and a lower plate arranged in parallel,

[0035] The upper plate has an upper substrate, and an upper hydrophobic layer is arranged on the lower bottom surface of the upper substrate,

[0036] The lower plate has a lower substrate, and the lower plate has a lower substrate, and the lower substrate has a digital microfluidic chip electrode layer, and an insulating medium layer is arranged on the digital microfluidic chip electrode layer, and a lower hydrophobic layer is arranged on the insulating medium layer,

[0037] The electrode layer of the digital microfluidic chip comprises a plurality of liquid storage areas, a plurality of reaction areas and at least one waste liquid area, and each of the plurality of liquid storage areas, the plurality of reaction areas and the at least one waste liquid area is provided with a corresponding electrode,

[0038] The electrodes corresponding to the plurality of liquid storage areas are connected to the electrodes corresponding to the plurality of reaction areas through a plurality of first electrodes, and the liquid reaction materials corresponding to the plurality of liquid storage areas are transported to the plurality of reaction areas through the alternating on-off of the electrodes corresponding to the plurality of liquid storage areas, the plurality of first electrodes and the electrodes corresponding to the plurality of reaction areas; each reaction area has a magnetic bead positioning area and a plurality of second electrodes surrounding the magnetic bead positioning area, and the liquid reaction materials corresponding to the reaction area are transported to the magnetic bead positioning area through the alternating on-off of the electrodes corresponding to the reaction area and the plurality of second electrodes, and the liquid reaction materials from the plurality of liquid storage areas are uniformly mixed with the magnetic bead liquid materials corresponding to the magnetic bead positioning area and react; the electrodes corresponding to the plurality of reaction areas are connected to the electrode corresponding to the waste liquid area through a plurality of third electrodes, and the used liquid materials after the reaction of the plurality of reaction areas are transported to the waste liquid area through the alternating on-off of the electrodes corresponding to the plurality of reaction areas, the plurality of third electrodes and the electrode corresponding to the at least one waste liquid area;

[0039] The upper plate and the lower plate are arranged in parallel, and the upper hydrophobic layer and the lower hydrophobic layer form a space for liquid flow, and the upper hydrophobic layer and the lower hydrophobic layer are in contact with the liquid.

[0040] Further preferably, the material of the substrate is a light-transmissive conductive material (such as ITO glass), and the material of the upper hydrophobic layer is a hydrophobic material (such as Teflon material) with a contact angle greater than 100 degrees.

[0041] Further preferably, the lower substrate is a printed circuit board, glass, silicon wafer or flexible material, the electrode layer of the digital microfluidic chip is a gold-plated copper electrode layer, a metal conductive electrode layer or a conductive polymer electrode layer, the material of the lower hydrophobic layer is a hydrophobic material (such as Teflon material) with a contact angle greater than 100 degrees, and the material of the insulating medium layer is preferably SU-8 photoresist.

[0042] The present application has the following advantages:

[0043] 1. The present application can provide an integrated solution from writing to reading of DNA information storage.

[0044] 2. The present application adopts a biological enzyme-catalyzed DNA synthesis method, which has the advantages of high efficiency, high fidelity, low cost and long fragment DNA synthesis, and does not need to introduce a complex and toxic reagent system.

[0045] 3、The digital microfluidic chip has the advantages of low cost, easy processing, easy integration, high parallelism and full automation in addition to the advantages of small reaction volume, heat and mass transfer, high efficiency, short reaction time, etc. BRIEF DESCRIPTION OF DRAWINGS

[0046] Figure 1 A structure diagram of the digital microfluidic chip prepared for the embodiment 1 of the present application. DETAILED DESCRIPTION

[0047] The technical solutions of the present application are further described and explained in detail through specific embodiments combined with the drawings.

[0048] Embodiment 1

[0049] As shown in the drawings, Figure 1 the digital microfluidic chip has a parallelly arranged upper plate and a lower plate,

[0050] The upper plate has an upper substrate, and an upper hydrophobic layer is arranged on the lower bottom surface of the upper substrate, the upper substrate is ITO glass, the upper hydrophobic layer is a Teflon material layer, the size of the upper plate is 76mm×68mm×1.1mm, and the square resistance is 23.1Ω,

[0051] The lower plate has a lower substrate, and the lower plate has a lower substrate, and a digital microfluidic chip electrode layer is arranged on the lower substrate, an insulating medium layer is arranged on the digital microfluidic chip electrode layer, and a lower hydrophobic layer is arranged on the insulating medium layer, the lower substrate is a printed circuit board (PCB), the digital microfluidic chip electrode layer is a gold-plated copper electrode layer, the material of the insulating medium layer is SU-8 photoresist, the lower hydrophobic layer is a Teflon material layer, and the size of the lower plate is 100mm×75mm×1.6mm.

[0052] The upper plate and the lower plate are arranged in parallel, a space for liquid flow is formed between the upper hydrophobic layer and the lower hydrophobic layer, and the upper hydrophobic layer and the lower hydrophobic layer both contact the liquid;

[0053] The digital microfluidic chip electrode layer includes six liquid storage areas 1, two reaction areas 2 and a waste liquid area 3, and each of the six liquid storage areas 1, the two reaction areas 2 and the waste liquid area 3 is provided with a corresponding electrode,

[0054] The electrodes corresponding to the six liquid storage areas 1 are connected to the electrodes corresponding to the two reaction areas 2 through the first electrodes 11, and the liquid reaction materials corresponding to the six liquid storage areas 1 are transported to the two reaction areas 2 through the electrodes corresponding to the six liquid storage areas 1, the first electrodes 11 and the electrodes corresponding to the two reaction areas 2; each reaction area 2 has a magnetic bead positioning area and a plurality of second electrodes 21 surrounding the magnetic bead positioning area, and the liquid reaction materials corresponding to the reaction area 2 are transported to the magnetic bead positioning area through the electrodes corresponding to the reaction area 2 and the second electrodes 21, and the liquid reaction materials from the six liquid storage areas 1 are uniformly mixed with the magnetic bead liquid materials corresponding to the magnetic bead positioning area and reacted through the alternation of the electrodes corresponding to the reaction area 2 and the second electrodes 21; the electrodes corresponding to the two reaction areas 2 are connected to the electrodes corresponding to the waste liquid area 3 through the third electrodes 31, and the used liquid materials after the reaction of the two reaction areas 2 are transported to the waste liquid area 3 through the electrodes corresponding to the two reaction areas 2, the third electrodes 31 and the electrodes corresponding to the waste liquid area 3.

[0055] The manufacturing of the digital microfluidic chip includes the following steps:

[0056] I. Manufacturing the upper plate

[0057] (1) Cleaning: The ITO glass is cleaned with 10% glass cleaning solution in a 65°C water bath for 40 minutes, and then ultrasonically cleaned with ultrapure water in a 65°C water bath for 15 minutes, repeated twice.

[0058] (2) Drying: The cleaned ITO glass is dried with nitrogen, and then placed in an oven with a temperature setting of 125°C for 40 minutes to obtain the upper substrate.

[0059] (3) Preparing the upper hydrophobic layer:

[0060] a. Surface activation: The upper substrate is placed in the chamber of the plasma cleaner for surface activation, with an oxygen pressure of 600 Torr in the chamber and an activation time of 30 seconds.

[0061] b. Spin coating Teflon material: The upper substrate is placed on the chuck of the spin coater, and an appropriate amount of oil-based Teflon with a concentration of 1% (w / w) is poured onto the surface of the upper substrate. The spin coating parameters are set as follows: rotation speed 4000 rpm, acceleration 200 rpm / s, and spin coating time 60 seconds.

[0062] c. Post-baking: The upper substrate with the spin-coated Teflon is placed on a hot plate, and the temperature of the hot plate is set to 200°C for 15 minutes. Thus, the manufacturing of the upper plate of the digital microfluidic chip is completed.

[0063] II. Manufacturing the lower plate

[0064] (1) Design: use Auto CAD 2015 software to draw design drawings, and then use Altium Designer 2020 software to design the circuit board. There are 98 pin electrodes and 96 driving electrodes on the lower plate. The driving electrodes are located in the center of the digital microfluidic chip, connected one by one by the lead wire, and two pin electrodes are left empty.

[0065] (2) Plate making: the circuit board design is handed over to the circuit board manufacturer for plate making, using FR-4 board material type, outer copper thickness 1 ounce, surface gold plating 1 ounce.

[0066] (3) Cleaning: ultrasonic cleaning of PCB in 65℃ water bath for 15min, repeated 2 times.

[0067] (4) Drying: use nitrogen to dry the cleaned PCB, dry the PCB at 200℃ for 15min using a heating table, and obtain the lower substrate.

[0068] (5) Making insulating medium layer:

[0069] a, uniform glue: first use Kapton high temperature resistant tape to protect the pin electrodes on both sides of the above lower substrate, pour a proper amount of SU-82015 negative photoresist on the surface of the PCB on the glue uniformity instrument, set the glue uniformity parameters, rotation speed 4000rpm, acceleration 200rpm / s, spin 60s.

[0070] b, pre-baking: place the spin-coated lower substrate on the heating plate, set the heating plate temperature to 65℃, and heat for 15min.

[0071] c, exposure: place the cooled lower substrate on the film holder of the photoetching machine, 365nm ultraviolet light, light power ~ 10mW / cm2, general exposure 15s.

[0072] d, post-baking: place the exposed lower substrate into the oven, set the temperature to 200℃, and dry for 40min to obtain the lower substrate with insulating medium layer.

[0073] (6) Making lower hydrophobic layer:

[0074] a, surface activation: place the lower substrate with insulating medium layer obtained above into the plasma cleaning instrument chamber for surface activation, oxygen pressure in the chamber is 600Torr, activation time is 30s.

[0075] b, spin-coating Teflon material: after taking out the material, place it on the chuck of the glue uniformity machine, pour a proper amount of oil-based Teflon with a concentration of 1%(w / w) on its surface, set the glue uniformity parameters, rotation speed 4000rpm, acceleration 200rpm / s, spin 60s.

[0076] c. Post-baking: the lower substrate on which the Teflon material was spin-coated was placed on a hot plate, the temperature of the hot plate was set to 200°C, and the heating time was 15 min. Thus, the fabrication of the lower plate of the digital microfluidic chip was completed.

[0077] Example 2

[0078] On a DNA information storage platform, the digital microfluidic chip prepared in Example 1 was applied to automatic in-situ DNA information storage and reading. The DNA information storage platform comprises a digital microfluidic chip control module, a permanent magnet mechanical movement module, a photomultiplier tube (PMT) chemiluminescence detection module, a central control communication module, a power module, and an upper computer and user interaction software. The digital microfluidic chip control module is mainly used to automatically control the movement, generation and splitting of droplets on the digital microfluidic chip, and is the basic platform for realizing DNA information storage; the permanent magnet mechanical movement module is mainly used for the magnetic separation process of magnetic bead droplets, and the module can move up and down mechanically and can perform magnetic separation process on the magnetic bead droplets at a specified position; the PMT chemiluminescence detection module is mainly used for the realization of DNA sequencing in DNA information storage, and can detect the chemiluminescence signal in the sample pyrophosphate sequencing process; the central control communication module is mainly used for information uplink and downlink between the upper computer and signal communication and control between each module; the power module is used to provide power supply for all modules; the upper computer and its software are used for interaction with the user, and the software is used to input the information data required to be stored and output the information data stored by DNA.

[0079] The automatic in-situ DNA information storage and reading method based on the above hardware comprises the following steps:

[0080] (1) converting the data file required to be stored into a ternary number through information exchange code, and in this embodiment, "XM" is selected for coding;

[0081] (2) coding the ternary number into a corresponding DNA sequence according to the above coding table for conversion between the ternary number and DNA bases,

[0082] The coding table for conversion is implemented according to the conversion between A, T, C and G, the four bases in the DNA sequence, and the coding of 0, 1 and 2 of the ternary number, wherein,

[0083] A→C, T→G, C→A and G→T are all converted to 0;

[0084] A→T, T→C, C→G and G→A are all converted to 1;

[0085] A→G, T→A, C→T and G→C are all converted to 2;

[0086] A→C means that the next position of A in the DNA sequence is C,

[0087] T→G means that the next position of T in the DNA sequence is G,

[0088] C→A means that the next position of C in the DNA sequence is A,

[0089] G→T means that the next position of G in the DNA sequence is T,

[0090] A→T means that the next position of A in the DNA sequence is T,

[0091] T→C means that the next position of T in the DNA sequence is C,

[0092] C→G means that the next position of C in the DNA sequence is G,

[0093] G→A means that the next position of G in the DNA sequence is A,

[0094] A→G means that the next position of A in the DNA sequence is G,

[0095] T→A means that the next position of T in the DNA sequence is A,

[0096] C→T means that the next position of C in the DNA sequence is T,

[0097] G→C means that the next position of G in the DNA sequence is C,

[0098] For example, as shown in Table 1:

[0099] Table 1 Encoding and decoding table

[0100]

[0101] According to the above encoding and decoding table, the present embodiment converts “XM” into a ternary number “02120110” and a DNA sequence “g*TATACGAC”, and then synthesizes according to the sequence;

[0102] (3) On a digital microfluidic chip, a DNA synthesis method based on terminal transferase (TdT) is used to synthesize the corresponding DNA sequence encoded in step (2) to store the information of the data file; the specific process is as follows:

[0103] A, preparation of solid phase carrier:

[0104] a. Streptavidin magnetic beads washing: In a centrifuge tube, take 20 μL streptavidin (SA) modified magnetic beads with a concentration of 10 mg / mL, mix with 180 μL magnetic bead washing buffer, place the centrifuge tube on the magnetic stand, stand for 3 min, discard the supernatant, and repeat this step 3 times.

[0105] b. Coupling of initiator sequence: In the above centrifuge tube, resuspend by adding 197 μL magnetic bead washing buffer, then add 3 μL Biotin-Initiator solution with a concentration of 100 μM, carry out the binding reaction of biotin and streptavidin, place in a rotary incubator, and incubate for 30 min.

[0106] c. Initiator magnetic bead washing: Wash the incubated magnetic beads with 200 μL ultrapure water 3 times, and then resuspend with 20 μL ultrapure water.

[0107] B. Buffer preparation:

[0108] a. Preparation of enzyme premix: Take a centrifuge tube, add 8.4 μL deionized water, 8 μL 5×TdT Co 2+ buffer, 2 μL TdT with a concentration of 20 U / μL, and 1.6 μL Triton X-100 solution with a concentration of 0.2%, and mix well by shaking.

[0109] b. Preparation of deoxynucleotide monomer (dNTP) premix: Take a centrifuge tube, add 16 μL deionized water, 4 μL deoxythymine nucleotide (T) with a concentration of 20 mM, and mix well by shaking to obtain a T base premix. Prepare a deoxycytine nucleotide (C) premix in the same way. Take a centrifuge tube, add 16 μL deionized water, 4 μL deoxyadenine nucleotide (A) with a concentration of 10 mM, to obtain an A base premix, and prepare a deoxyguanine nucleotide (G) premix in the same way.

[0110] c. Preparation of PBS termination solution: Take a centrifuge tube, mix 19 μL ultrapure water with 1 μL 20×PBS buffer, dilute to 1×PBS buffer, and prepare for use.

[0111] C. DNA synthesis:

[0112] a, loading: the lower plate of the digital microfluidic chip is fixed on the DNA information storage platform, the electrodes corresponding to the four reservoirs 1 of A, T, C and G dNTPs on the digital microfluidic chip are powered on, and 20 μL of the prepared four kinds of dNTP premix solution is added respectively, the electrode position corresponding to the enzyme solution of one reservoir 1 on the digital microfluidic chip is powered on, and 20 μL of the prepared enzyme premix solution is added respectively, the electrode position corresponding to the termination solution of one reservoir 1 on the digital microfluidic chip is powered on, and 20 μL of the prepared PBS termination solution is added respectively, 2 μL of the prepared starting linker magnetic bead solution is added dropwise in the reaction area 2, the upper plate is covered, and the upper plate is grounded. The instrument platform control realizes the pressing of the needle, the pin electrode of the lower plate is aligned with the spring needle electrode array, and the digital microfluidic chip is fixed. After the completion of the fixing, the dimethyl silicone oil is filled between the upper and lower plates.

[0113] b, single round synthesis standard procedure: the whole process is controlled by the DNA information storage platform: first, the magnetic column under the digital microfluidic chip is raised, the magnetic bead solution is enriched, and the alternating on-off of a plurality of third electrodes 31 is controlled to remove the supernatant from the reaction area 2 to the waste liquid area 3; then, the electrodes corresponding to the T base reservoir 1 and the corresponding first electrode 11 are sequentially powered on and off to separate 2 μL of the base solution mixed with the magnetic bead solution; then, the electrodes corresponding to the enzyme solution reservoir 1 and the corresponding first electrode 11 are sequentially powered on and off to separate 2 μL of the enzyme solution mixed with the magnetic bead solution; then, the second electrode 21 around the magnetic bead solution is powered on to circulate the magnetic bead solution, and the T base synthesis reaction is started, with a reaction time of 1 min; finally, the electrodes corresponding to the termination solution reservoir 1 and the corresponding first electrode 11 are sequentially powered on and off to separate 2 μL of the PBS termination solution mixed with the magnetic bead solution, and the single round synthesis reaction is stopped.

[0114] c, repeated cycle synthesis: repeat the above single round synthesis reaction, and according to the sequence of DNA sequence "T→A→T→A→C→G→A→C", repeat the above single round synthesis standard procedure (replace different base solution types) until eight rounds of synthesis are completed.

[0115] d, recovery: the needle is raised, and the unused solutions on the reservoir 1 of A, T, C and G four dNTPs, the reservoir 1 of enzyme solution and the reservoir 1 of termination solution are taken out and recovered respectively, and the waste liquid in the waste liquid area 3 is removed (optional).

[0116] (4) On the digital microfluidic chip, the splinted end ligation reaction based on T4 DNA Ligase and the direct pretreatment of the sample to make it available for in situ pyrophosphorolysis sequencing are adopted. Hereinafter, the splinted end ligation reaction and the sequencing pretreatment steps for the DNA sequence "g*TATACGAC" on the DNA information storage chip platform are described.

[0117] A, buffer preparation:

[0118] a, preparation of ligation cleaning solution: take 1 centrifuge tube, add 18 μL ultrapure water and 2 μL 10x Ligase buffer and mix well, dilute to 1x Ligase buffer for standby.

[0119] b, preparation of ligation reaction solution: take 1 centrifuge tube, add 12 μL ultrapure water, 2 μL 10x Ligase buffer, 1 μL Splint-G splint chain solution with a concentration of 100 mM and 1 μL 5Phos-Tail Adapter tail adapter solution with a concentration of 100 mM, mix well for standby.

[0120] c, preparation of sequencing pretreatment cleaning solution: take 1 centrifuge tube, add 100 mM Tris-acetate (pH 7.6), 0.5 mM ethylenediaminetetraacetic acid, 5 mM magnesium acetate and 0.01% (v / v) Tween 20, mix well for standby.

[0121] d, preparation of single-strand binding protein reaction solution: take 1 centrifuge tube, add 2.4 μL single-strand binding protein with a concentration of 5 mg / mL and 17.6 μL sequencing pretreatment cleaning solution, mix well for standby.

[0122] B, splinted end ligation reaction and sequencing pretreatment:

[0123] a, sample loading: power on the electrode position corresponding to the four reservoir area 1 on the chip, and add 20 μL ligation cleaning solution, ligation reaction solution, sequencing pretreatment cleaning solution and single-strand binding protein reaction solution respectively, the instrument platform controls the needle to press down, so that the lower plate pin electrode and the spring needle electrode array are aligned, and dimethyl silicone oil is appropriately supplemented between the upper and lower electrode plates.

[0124] b. Clamping end ligation reaction: The whole process is controlled by the DNA information storage platform described above. First, the magnetic column under the instrument chip is raised to enrich the magnetic bead solution, and the corresponding electrodes of the reaction area 2 and the corresponding several third electrodes 31 are controlled to remove the supernatant to the waste liquid area 3; then, the voltage is applied to the electrodes corresponding to the ligation cleaning solution storage area 1 and the corresponding several first electrodes 11 in turn to perform alternating on-off, and 2 μL of ligation cleaning solution and magnetic bead solution are mixed uniformly, and this cleaning step is repeated twice; then, the voltage is applied to the electrodes corresponding to the ligation reaction solution storage area 1 and the corresponding several first electrodes 11 in turn to perform alternating on-off, and 2 μL of ligation reaction solution and magnetic bead solution are mixed uniformly; then, the voltage is applied to the several second electrodes 21 around the magnetic bead solution to circulate the magnetic bead solution, and the clamping end ligation reaction is started, with a reaction time of 5 min; finally, the voltage is applied to the electrodes corresponding to the sequencing pretreatment cleaning solution storage area 1 and the corresponding several first electrodes 11 in turn to perform alternating on-off, and 2 μL of sequencing pretreatment cleaning solution and magnetic bead solution are mixed uniformly, and this cleaning step is repeated twice.

[0125] c. Sequencing pretreatment: The whole process is controlled by the DNA information storage platform described above. First, the magnetic column under the instrument chip is raised to enrich the magnetic bead solution, and the corresponding electrodes of the reaction area 2 and the corresponding several third electrodes 31 are controlled to remove the supernatant to the waste liquid area 3; then, the voltage is applied to the electrodes corresponding to the single-strand binding protein reaction solution storage area 1 and the corresponding several first electrodes 11 in turn to perform alternating on-off, and 2 μL of single-strand binding protein reaction solution and magnetic bead solution are mixed uniformly; then, the voltage is applied to the several second electrodes 21 around the magnetic bead solution to circulate the magnetic bead solution, and the sequencing pretreatment is started, with a reaction time of 10 min; finally, the voltage is applied to the electrodes corresponding to the sequencing pretreatment cleaning solution storage area 1 and the corresponding several first electrodes 11 in turn to perform alternating on-off, and 2 μL of sequencing pretreatment cleaning solution and magnetic bead solution are mixed uniformly, and this cleaning step is repeated twice.

[0126] d. Recovery: The upper needle is raised to take out and recover the unused solutions in the four storage areas 1 of the ligation cleaning solution, the ligation reaction solution, the sequencing pretreatment cleaning solution, and the single-strand binding protein reaction solution, respectively.

[0127] (5) The DNA sequence synthesized in step (3) is sequenced by the pyrophosphate DNA sequencing method on the digital microfluidic chip to read the information of the data file; the specific process is as follows:

[0128] A. Buffer preparation:

[0129] a. Preparation of sequencing washing solution: Take 1 centrifuge tube, add 100 mM Tris-acetate (pH 7.6), 0.5 mM ethylenediaminetetraacetic acid, 5 mM magnesium acetate, and 0.01% (v / v) Tween 20, mix well for standby use.

[0130] b. Preparation of 2x sequencing washing solution: Take 1 centrifuge tube, add 200 mM Tris-acetate (pH 7.6), 1 mM ethylenediaminetetraacetic acid, 10 mM magnesium acetate, and 0.02% (v / v) Tween 20, mix well for standby use.

[0131] c. Preparation of enzyme premix solution: Take 1 centrifuge tube, add 20 mU / μL adenine nucleotide triphosphate (ATP) sulfating enzyme, 0.12 μg / μL luciferase, 0.6 μg / μL luciferin, and 0.5 U / μL Klenow fragment polymerase, and add half of the final volume of 2x sequencing washing solution, and the rest is supplemented with ultrapure water, mix well for standby use.

[0132] d. Preparation of dNTP substrate premix solution: Take 4 centrifuge tubes, respectively prepare A, T, C, and G four dNTP substrate premix solutions, respectively add 20 μM single dNTPs (A base uses dATP-α-S, T, C, G base uses dTTP, dCTP, dGTP), 33.3 μM adenosine 5'-phosphosulfate (APS), 1 mM dithiothreitol (DTT), and 120 ng / μL single-stranded binding protein, and add half of the final volume of 2x sequencing washing solution, and the rest is supplemented with ultrapure water, mix well for standby use.

[0133] B. DNA sequencing:

[0134] a. Loading: Power on the 6 reservoir area 1 electrode positions on the chip, and respectively add 20 μL of sequencing washing solution, enzyme premix solution, and four dNTP substrate premix solutions, and the instrument platform controls the needle to press down to align the lower plate pin electrode with the spring needle electrode array, and appropriately supplement dimethyl silicone oil between the upper and lower electrode plates.

[0135] b. Single round sequencing standard procedure: The whole process is controlled by the DNA information storage instrument platform. First, the magnetic column under the instrument chip is raised to enrich the magnetic bead solution, and the corresponding electrodes of the reaction area 2 of the magnetic bead solution and the corresponding several third electrodes 31 are controlled to remove the supernatant to the waste liquid area 3; then, the electrodes corresponding to the A base substrate premix solution storage area 1 and the corresponding several first electrodes 11 are sequentially applied with voltage for alternating on-off to separate 2 μL of A base substrate premix solution and mix with the magnetic bead solution; then, the electrodes corresponding to the enzyme premix solution storage area 1 and the corresponding several first electrodes 11 are sequentially applied with voltage for alternating on-off to separate 2 μL of enzyme premix solution and mix with the magnetic bead solution; then, the several second electrodes 21 of the reaction area 2 of the magnetic bead solution are applied with voltage for alternating on-off to circulate the magnetic bead solution, start the A base sequencing reaction, the reaction time is 1 min, then directly detect the chemiluminescence signal by PMT, if the signal exceeds the threshold value, it is a positive signal, indicating that the sequencing result of the site is "T base", the sequencing of the site is ended, if the signal does not exceed the threshold value, it is a negative signal, indicating that the sequencing result of the site is not "T base", continue the sequencing of the site; finally, the corresponding electrodes of the sequencing cleaning solution storage area 1 and the corresponding several first electrodes 11 are sequentially applied with voltage for alternating on-off to separate 2 μL of sequencing cleaning solution and mix with the magnetic bead solution, and the cleaning step is repeated for 3 times.

[0136] c. Repeat cycle sequencing: repeat the above single round sequencing reaction, and the sequencing is cycled in the order of "A→T→C→G". The above single round sequencing standard procedure is repeated (replace different base solution types) until the sequencing of the eight sites is completed.

[0137] d. Recovery: raise the thimble to take out and recover the unused solutions on the sequencing cleaning solution, enzyme premix solution and four dNTP substrate premix solution storage areas 1, at the same time, take out the chip, clean and dry it according to the foregoing method, and the chip can be repeatedly used.

[0138] (6) Decode the sequencing signal obtained in step (5), decode according to the above table 1, convert "g*TATACGAC" by the decoding mode of "DNA sequence-ternary number-character", sequentially obtain ternary number "02120110" and character information "XM", and complete the whole process of DNA-based information encoding, writing, storage, reading and decoding.

[0139] The above is only a preferred embodiment of the present application, and therefore cannot limit the scope of the present application. Equivalent changes and modifications made in accordance with the scope and content of the present patent should still be within the scope of the present application.

Claims

1. An automated in situ DNA information storage and reading method, characterized by: The method comprises the following steps: (1) converting the data file to be stored into ternary numbers through information exchange code; (2) encoding the ternary numbers into corresponding DNA sequences according to the correspondence coding table between the ternary numbers and DNA base transformation; (3) synthesizing the corresponding DNA sequence encoded in step (2) on a digital microfluidic chip by using a biological enzyme-catalyzed DNA synthesis method, a chemical phosphoramidite DNA synthesis method or an enzyme ligation DNA synthesis method to store the information of the data file; (4) sequencing the DNA sequence synthesized in step (3) on the digital microfluidic chip by using a DNA sequencing method to read the information of the data file; (5) decoding the information read in step (4) according to the correspondence coding table; The digital microfluidic chip has an upper substrate and a lower substrate arranged in parallel, the upper substrate has an upper base, and an upper hydrophobic layer is arranged on the lower bottom surface of the upper base, the lower substrate has a lower base, and a digital microfluidic chip electrode layer is arranged on the lower base, the digital microfluidic chip electrode layer is provided with an insulating medium layer, and a lower hydrophobic layer is arranged on the insulating medium layer, the digital microfluidic chip electrode layer comprises a plurality of liquid storage areas, a plurality of reaction areas and at least one waste liquid area, and each of the plurality of liquid storage areas, the plurality of reaction areas and the at least one waste liquid area is provided with a corresponding electrode, the electrodes corresponding to the plurality of liquid storage areas are connected to the electrodes corresponding to the plurality of reaction areas through a plurality of first electrodes, and the liquid reaction materials in the plurality of liquid storage areas are transported to the plurality of reaction areas through the alternating on-off of the electrodes corresponding to the plurality of liquid storage areas, the plurality of first electrodes and the electrodes corresponding to the plurality of reaction areas; each reaction area has a magnetic bead positioning area and a plurality of second electrodes surrounding the magnetic bead positioning area, and the liquid reaction materials in the reaction area are transported to the magnetic bead positioning area through the alternating on-off of the electrodes corresponding to the reaction area and the plurality of second electrodes, and the liquid reaction materials from the plurality of liquid storage areas are uniformly mixed with the magnetic bead liquid materials in the magnetic bead positioning area and react; the electrodes corresponding to the plurality of reaction areas are connected to the electrode corresponding to the waste liquid area through a plurality of third electrodes, and the unused liquid materials after the reaction of the plurality of reaction areas are transported to the waste liquid area through the alternating on-off of the electrodes corresponding to the plurality of reaction areas, the plurality of third electrodes and the electrode corresponding to the at least one waste liquid area; The upper substrate and the lower substrate are arranged in parallel, and the space for liquid flow is formed between the upper hydrophobic layer and the lower hydrophobic layer, and the upper hydrophobic layer and the lower hydrophobic layer both contact the liquid.

2. The automated in situ DNA information storage and readout method of claim 1, wherein: The information exchange code includes text, pictures, music and videos.

3. The automated in situ DNA information storage and readout method of claim 2, wherein: The text includes ASCII and GBK, the pictures include JPG and BMP, the music includes MAV and MP3, and the video includes MPEG and H.26X.

4. The automated in situ DNA information storage and readout method of claim 1, wherein: The correspondence coding table realizes the coding of 0, 1 and 2 of ternary numbers according to the transformation between A, T, C and G in the DNA sequence, wherein, A→C, T→G, C→A and G→T are all converted into 0; A→T, T→C, C→G and G→A are all converted into 1; A→G, T→A, C→T and G→C are all converted into 2; A→C represents the case that the next position of A in the DNA sequence is C, T→G represents the case that the next position of T in the DNA sequence is G, C→A represents the case that the next position of C in the DNA sequence is A, G→T represents the case that the next position of G in the DNA sequence is T, A→T represents the case that the next position of A in the DNA sequence is T, T→C represents the case that the next position of T in the DNA sequence is C, C→G represents the case that the next position of C in the DNA sequence is G, G→A represents the case that the next position of G in the DNA sequence is A, A→G represents the case that the next position of A in the DNA sequence is G, T→A represents the case that the next position of T in the DNA sequence is A, C→T represents the case that the next position of C in the DNA sequence is T, G→C represents the case that the next position of G in the DNA sequence is C.

5. The automated in situ DNA information storage and readout method of claim 1, wherein: The biological enzymatic DNA synthesis method is an endonuclease-based enzymatic DNA synthesis method, a polynucleotide phosphorylase-based DNA chemical synthesis method or a T4 RNA ligase-based DNA synthesis method.

6. The automated in situ DNA information storage and readout method of claim 1, wherein: The DNA sequencing method is a pyrophosphate DNA sequencing method, a Sanger sequencing method, a second-generation sequencing method or a nanopore third-generation sequencing method.

7. The automated in situ DNA information storage and readout method of claim 1, wherein: The material of the upper substrate is a light-transmissive conductive material, and the material of the upper hydrophobic layer is a hydrophobic material with a contact angle greater than 100 degrees.

8. The automated in situ DNA information storage and readout method of claim 1, wherein: The lower substrate is a printed circuit board, glass, a silicon wafer or a flexible material, the digital microfluidic chip electrode layer is a gold-plated copper electrode layer, a metal conductive electrode layer or a conductive polymer electrode layer, and the material of the lower hydrophobic layer is a hydrophobic material with a contact angle greater than 100 degrees.

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