Patent Description:
With the development of human society, the accumulated amount of information shows an explosive growth trend. It has been predicted in IDC's report "<NPL> the total amount of global data will exceed <NUM> ZB! Moreover, the amount of global data is still growing rapidly at a rate of <NUM>% per year, and a large amount of valid data is being lost. Data storage is a problem all over the world. The commonly used storage media at present, such as optical disks and hard disks, have disadvantages such as low storage capacity, large volume, high cost of maintenance and short storage time (~<NUM> years). In order to solve these problems fundamentally, it is necessary to develop a novel information storage medium as soon as possible.

DNA-based storage is a future-focused, subversive information storage technology. The use of DNA as an information storage medium has many advantages such as small volume, large storage capacity, strong stability and low cost of maintenance. Theoretically, <NUM> gram of DNA can store thousands of terabytes of data, from which it is estimated that the storage of all the existing information of human beings including books, files, videos, etc. can be achieved by using only hundreds of kilograms of DNA, and the storage time can be up to thousands of years under normal conditions. Therefore, those information that is not commonly used but needs long-term preservation, such as government documents and historical files, etc., is especially suitable for DNA-based storage.

Although DNA-based storage has many superior advantages as compared with the existing storage, there are some technical barriers that hinder its development, such as the inability to reuse synthetic DNA oligo fragments, high cost of DNA synthesis, complex design and poor flexibility, etc., resulting in difficulties in large-scale promotion and application of the existing DNA-based storage technology. Therefore, it is necessary to start from the design of basic information-constituting unit to optimize the coding design of DNA-based storage, thereby reducing costs and improving efficiency and convenience.

The following documents are also considered as the prior art but are apparently distinguished from the present invention:.

It is an object of the present invention to provide a method for encoding and storing text information by using DNA as a storage medium, and a decoding method therefor and application thereof.

The method for storing text information provided by the present invention generally comprises: firstly, encoding a character into a computer binary digit by encoding, and then converting the binary digit into a DNA sequence by transcoding; and secondly, artificially synthesizing the DNA sequence encoding the character information and locating the character by a designed ligation adapter to assemble the DNA sequences encoding the characters in a preset order. Alternatively, the assembled DNA sequences can be further assembled into a longer DNA sequence as needed.

In the method for storing text information of the present invention, each character can be used repeatedly, and by changing the adapter, can be used for storing any information, the principle of which is the same as that of the "movable-type printing" strategy. The DNA which has stored text information can be preserved under appropriate conditions. When the stored information needs to be read, the stored character information can be obtained by sequencing the DNA sequence followed by decoding with a computer (as shown in <FIG>). The method provided by the present invention has the advantages of small storage volume, large storage capacity, strong stability and low cost of maintenance, etc by using DNA as a storage medium.

Specifically, the technical object of the present invention can be achieved by the following aspects:
In a first aspect, the present invention provides a method for storing text information by using DNA as a storage medium, comprising the steps of:.

In an alternative particular embodiment, the encoding is Unicode-ucs2 encoding; that is, each Chinese character is encoded by a hexadecimal digit, for example, the corresponding Unicode code of the character "<IMG>" is U+<NUM>; each <NUM>-bit hexadecimal digit is converted into a <NUM>-bit binary digit, for example, <NUM> is converted into <NUM> and <NUM> is converted into <NUM>, and thus the character "<IMG>" is converted into a binary digit <NUM>; preferably, each <NUM>-bit binary digit produce a <NUM>-bit Hamming code for verification, and thus the Hamming codes of the character "<IMG>" are <NUM> and <NUM> respectively. Finally, a complete binary code of the character "<IMG>" can be obtained, that is <NUM>.

In an optional particular embodiment, the transcoding is performed according to the principle that the binary digit <NUM> is converted into G or T and the binary digit <NUM> is converted into C or A so as to convert the binary digit encoding a character into a DNA sequence.

Preferably, one Chinese character is encoded into <NUM> bases.

Preferably, the sequence design is controlled by considering one or more of parameters including GC content, secondary structure and base repetition rate of the DNA sequence; for example, preferably, the DNA sequence is designed such that the GC content thereof is <NUM>-<NUM>%, preferably <NUM>%; preferably, the DNA sequence is designed to avoid the formation of secondary structure; preferably, the DNA sequence is designed such that no more than <NUM> consecutive single bases are present therein. Taking the character "<IMG>" as an example, it is finally converted into a DNA sequence TAGCTATAGGCTTGCATAGCACCG.

Both the DNA sequence and the ligation adapter sequence in the present invention are obtained by de novo chemically synthesizing the forward and reverse strands and allowing them to anneal to form a double-stranded structure.

In an optional particular embodiment, a complementary locating base protrudes from both the DNA sequence fragment and the ligation adapter. The directional ligation of the DNA sequence to the ligation adapter is achieved via the complementary bases (i.e. , "locating base") respectively protruding from the DNA sequence and the ligation adapter. By designing the ligation adapter, DNA sequence fragments encoding various characters can be ligated in the desired character order.

In an optional particular embodiment, the ligation adapter comprises an upstream adapter and a downstream adapter; ligation adapters with the same DNA sequence but the different overhanging locating bases will linked to the upstream and downstream of two DNA fragments respectively, and the resulted two DNA fragments can be ligated by the ligation adapters by using a conventional molecular biology method, preferably by PCA, GoldenGate, etc. (as shown in <FIG>).

For example, one base protrudes from each end of a DNA fragment respectively, such as a base "A" protrudes from the sense strand and a base "G" protrudes from the antisense strand of the DNA fragment at <NUM>'-end, in which case, a base "T" should protrude from the antisense strand of the corresponding upstream adapter and a base "C" should protrude from the sense strand of the downstream adapter such that the directional ligation of the fragment to the adapter can be achieved by means of A/T and G/C pairing, that is, the adapter overhanging a "T" can only be linked at upstream of the DNA fragment and the adapter overhanging a "C" can only be linked at downstream of the DNA fragment. Similarly, the bases protruding from upstream and downstream of a DNA fragment may also be A/C, T/G and T/C, etc., in which case the corresponding bases protruding from the adapter become T/G, A/C and A/G, etc., correspondingly (as shown in <FIG>). Similarly, more than one unpaired base may protrude from each of the DNA fragment and the adapter. Similarly, the base may protrude from the DNA fragment and the adapter at the <NUM>'-end thereof. A base "G" may protrude from the sense strand of the DNA fragment at both <NUM>'-end and <NUM>'-end, in which case, a base "C" should protrude from the antisense strand of the corresponding upstream adapter at <NUM>'-end and the antisense strand of the downstream adapter at <NUM>'-end, also allowing the directional ligation of the fragment to the adapter. Similarly, the bases protruding from the sense strand of a DNA fragment at <NUM>'-end and <NUM>'-end may also be "C", "T" or "A", in which case the bases protruding from the adapter should become "G", "A" or "T", correspondingly. Similarly, "G", "C", "T" or "A" may protrude from the antisense strand of a DNA fragment, in which case "C", "G", "A" or "T" should protrude from the sense strand of the adapter, correspondingly (as shown in <FIG>). Similarly, more than one complementary base may protrude from each of the DNA fragment and the adapter.

Sequence of the ligation adapter can be automatically generated by a computer program. For example, a PCA adapter needs to have a length of more than <NUM> bp, a GC content of <NUM>%-<NUM>%, no secondary structure, no more than <NUM> consecutive bases, and no mismatch between the same set of adapters, etc.; a GoldenGate adapter consists of an enzymatic cleavage site sequence and its outer protective bases, and the difference in the <NUM> bp sticky ends resulting from enzyme restriction between the same set of adapters needs to be more than <NUM> bp (as shown in <FIG>). The <NUM>'-ends of the sense and antisense strands of the DNA fragment, the antisense strand of the upstream adapter, and the sense stand of the downstream adapter are phosphorylated. The <NUM>'-ends of the sense strand of the upstream adapter and the antisense strand of the downstream adapter are dephosphorylated to reduce the probability of self-linking and misligation of the adapters.

To the DNA sequences encoding the characters are respectively added the designed ligation adapters, though which locating is achieved; in a particular embodiment, by overlap extension PCR, individual DNA sequences comprising the encoding information of individual characters are ligated according to the character order of the information to be stored, and the ligated sequences can be further assembled into a longer DNA sequence; preferably, individual DNA sequences comprising the encoding information of individual characters are ligated by a method such as PCA or GoldenGate; preferably, the ligated DNA sequences are assembled by a Gibson method and the assembled DNA sequence which encodes the character information can be preserved under suitable storage conditions, for example, can be lyophilized for long-term storage at low temperatures.

In a particular embodiment, characters may be firstly assembled into a form of phrase or idiom, etc., such that the subsequent assembly becomes more convenient and efficient; for example, <NUM>-<NUM> characters may be assembled into a short sentence at once by using a method such as PCA or GoldenGate, etc., and then the short sentences can be further spliced into a long sentence, a paragraph or an article by using an assembly method such as Gibson assembly, etc..

Preferably, the assembled DNA sequence is cloned into a plasmid for storage; preferably, a step of verifying the correctness of the assembled DNA sequence by sequencing is also included prior to the storage.

In a second aspect, the present invention also provides a method for decoding the text information stored according to the method of the first aspect, comprising the steps of:.

When a mutation exists in the DNA sequence, it can be corrected by the Hamming code. For example, if the base at position <NUM> of the above DNA sequence is mutated from A to G, i.e. the DNA sequence becomes TGGCTATAGGCTTGCATAGCACCG, the corresponding binary digit will become <NUM>. It can be calculated according to the Hamming code verification principle that the base at position <NUM> is mutated, and thereby the binary digit will be corrected to <NUM> and the sequence can still be correctly decoded as "<IMG>".

Therefore, in particular embodiments, the decoding may further include the step of correcting mutations in the DNA sequence according to the Hamming code verification principle.

In a third aspect, the present invention provides use of the method for storing text information according to the first aspect and/or the method for decoding the stored text information according to the second aspect in the storage and/or reading of text information.

First, the method for storing text information according to the present invention has the advantages of small storage volume, large storage capacity, strong stability and low cost of maintenance, etc. by using DNA as a storage medium.

In addition, compared with other existing DNA storage methods, the present method is more suitable for storing text information, supporting text forms including characters of various countries including all Chinese characters, English letters, Japanese and Korean, etc., punctuation marks and mathematical symbols, etc.; has high encoding efficiency, wherein <NUM> Chinese characters can be encoded within <NUM> milliseconds; adopts a strategy similar to "movable-type printing", wherein the DNA fragments and adapters can be used repeatedly, resulting in lower cost of synthesis; the stored DNA sequence can be in a double-stranded closed circular conformation, which is more stable in storage; the stored DNA sequence can be verified by sequencing and the Hamming verification code can be added thereto, allowing any one mutation in every <NUM> bases, which results in a more fidelity; and the stored DNA sequence is a long double-stranded DNA, which is more easier for reading information.

The experimental procedures mentioned in the examples are conventional experimental methods unless otherwise specified; the reagents and consumables mentioned are conventional reagents and consumables unless otherwise specified. The synthetic oligos used in the experiments were diluted to <NUM> and the primers were diluted to <NUM> with sterile water.

The phrase for the assembly test: <IMG>.

Transcoding was performed according to the method of the present invention and DNA oligo sequences were synthesized; the PCA adapters were used for locating and ligating according to G/C and A/T base pairing manner at the upstream and downstream, respectively. The DNA oligo and primer sequences were shown in Table <NUM> below.

The forward and reverse oligos were each taken <NUM>µL for each character or adapter, mixed and annealed; the annealing procedure was: denatured at <NUM> for <NUM>, slowly cooled to <NUM> at <NUM>/sec, maintained at <NUM>.

To each character were added upstream and downstream adapters in order respectively for ligation; the ligation system was: <NUM>µL of T4 DNA ligase (Enzymatics), <NUM>µL of <NUM> × ligation buffer, <NUM>µL each of the annealed character, upstream adapter and downstream adapter, <NUM>µL of ddH<NUM>O; ligating at <NUM> overnight.

The ligation products were subjected to gel electrophoresis on <NUM>% PAGE gel at <NUM> V for <NUM> (the electrophoresis results were shown in <FIG>); the target bands (<NUM> bp in size, as indicated by the arrow in <FIG>) were cut off and purified: the cut gel was placed into a <NUM> tube with punctured bottom, which was then placed into a <NUM> tube, centrifuged at <NUM>,<NUM> rpm for <NUM>, <NUM>µL of <NUM> NaCl was added to the broken gel, shaken at <NUM> rpm at <NUM> for <NUM>; the broken gel and the liquid were transferred together into a filter column, centrifuged at <NUM>,<NUM> rpm for <NUM>, the filtrate was transferred into a <NUM> centrifuge tube, <NUM>µL of absolute ethanol was added, left for sinking at -<NUM> for <NUM>. Centrifuged at <NUM> rpm at <NUM> for <NUM>, discarded the supernatant, <NUM>µL of <NUM>% ethanol was added for washing the precipitate once, drawn off the supernatant, dried at <NUM> for <NUM>, and <NUM>µL of ddH<NUM>O was added to dissolve the DNA.

The characters were assembled into a short sentence by using a method of PCA. Step <NUM>: <NUM>µL of Ex Taq DNA polymerase (TAKARA), <NUM>µL of <NUM>×buffer, <NUM>µL of <NUM> dNTPs, <NUM> ng each of the ligated, cut and purified products of adapter <NUM>-U + <IMG> + adapter2-D, adapter2-U + <IMG> + adapter5-D, adapter5-U + <IMG> + adapter6-D, adapter6-U + <IMG> + adapter7-D, adapter7-U + <IMG> + adapter8-D, adapter8-U + <IMG> + adapter9-D and adapter9-U+ <IMG> + adapter10-D, adding water to <NUM>µL. <NUM> for <NUM>; <NUM> for <NUM> sec, <NUM> for <NUM> sec, cooled to <NUM> at <NUM>/sec, <NUM> for <NUM> sec, <NUM> for <NUM>, <NUM> cycles; <NUM> for <NUM>, maintained at <NUM>. Step <NUM>: <NUM>µL of Ex Taq DNA polymerase (TAKARA), <NUM>µL of <NUM>×buffer, <NUM>µL of <NUM> dNTPs, <NUM>µL of the PCR product of step <NUM>, <NUM>µL each of the primers <NUM>-F and St1-R, <NUM>µL of ddH<NUM>O. <NUM> for <NUM>; <NUM> for <NUM> sec, <NUM> for <NUM> sec, <NUM> for <NUM> sec, <NUM> cycles, <NUM> for <NUM>, maintained at <NUM>∘.

The PCR product was detected by electrophoresis: <NUM>µL of PCR product was used for electrophoresis detection. The electrophoresis was performed with <NUM>% agarose gel at <NUM> V for <NUM> (The electrophoresis result was shown in <FIG>. The PCR product was approximately <NUM> bp in size as indicated by the arrows).

The PCR product obtained in step <NUM> was purified by gel purification with a gel purification kit and the purified PCR product was cloned with a TA cloning kit (TAKARA).

Monoclones were selected from the TA cloning plate obtained in step <NUM>, incubated overnight, followed by plasmid extraction with a kit and identification by restriction digestion with the designed BssHII restriction site. The digestion system was: <NUM>µL of BssHII (NEB), <NUM>µL of CutSmart buffer, <NUM>µL of plasmid DNA and <NUM>µL of ddH<NUM>O. Digested at <NUM> for <NUM>. <NUM>µL of the digested product was used for electrophoresis with <NUM>% agarose gel at <NUM> V for <NUM> (the electrophoresis results were shown in <FIG> and the band with correct size was indicated by the arrow).

The correctly enzyme-digested plasmid was selected for sanger sequencing, and the plasmid with the correct assembly sequence was analyzed and obtained.

Claim 1:
A method for storing text information by using DNA as a storage medium, comprising the steps of:
(<NUM>) encoding a character into a computer binary digit by encoding;
(<NUM>) converting the computer binary digit encoding the character into a DNA sequence, which is represented by the four deoxyribonucleotides A, T, G, and C, by transcoding;
(<NUM>) synthesizing the DNA sequence encoding the character;
(<NUM>) locating the DNA sequence encoding the character by a designed ligation adapter, ligating individual DNA sequences encoding individual characters according to the order of characters of the information to be stored, followed by assembling and storing, wherein the ligation adapter includes an upstream adapter and a downstream adapter, and the <NUM>'-ends of the sense strand of the upstream adapter and the antisense strand of the downstream adapter are dephosphorylated.