Method for constructing DNA (Deoxyribose Nucleic Acid) type storage unit, attachment joint library and kit

Abstract

The invention discloses a method for constructing a DNA (Deoxyribonucleic Acid) type storage unit, a connection joint library and a kit. According to the method, on the basis of a DNA type storage thought, design of a DNA type library and construction of a DNA type physical library (including indexes, contents and verification information) are completed according to a specific principle. When a target file is stored, digital information is converted into quaternary DNA basic group information through a coding mapping mechanism, required types are called from a DNA type physical library, and construction of corresponding DNA type storage units is completed through necessary reaction, so that DNA type storage of the target file is realized. The DNA types can be repeatedly used, the data information writing cost is low, the single-time writing efficiency is high, the DNA storage connection scheme is flexible and changeable, researchers / demand users can select preferentially according to different file storage requirements and costs, and a customizable data storage kit is provided.

Description

Technical Field

[0001] The invention belongs to the field of DNA storage, and in particular relates to a method for constructing a DNA movable type storage unit, a connection joint library and a reagent kit. Background Art

[0002] Global data is growing explosively, and existing storage media can no longer meet the storage needs of massive amounts of data, and DNA storage technology is expected to break through this bottleneck. DNA storage technology uses DNA's natural information encoding and storage capabilities to convert 0-1 binary codes into DNA's A / T / C / G quaternary nucleotide codes, and then stores text, pictures, sound files and other data through artificial synthesis of specific nucleic acid sequences, and then uses sequencing and other decoding methods to read the data. Compared with existing silicon-based data information storage methods, this technology has the advantages of high data density, long preservation time, low energy consumption of supporting equipment, easy to carry, high transportation concealment, and easy multiple encryption. It is suitable for a variety of application scenarios such as large-scale cold data storage and data encryption transmission.

[0003] At present, the mainstream high-throughput DNA storage mostly adopts chemical synthesis methods in the data writing stage, which is costly and inefficient, and there is still a gap with practical application. The inventor first proposed the concept of "DNA movable type". Based on this concept, the data to be stored in the target file is split into multiple data elements, and the index / content information is annotated for each data element, corresponding to a specific double-stranded nucleic acid fragment in the corresponding movable type physical library. Subsequently, multiple content / index nucleic acid fragments are connected to form a DNA movable type unit, and multiple DNA movable type units constitute a DNA storage file that stores all the data elements to be stored.

[0004] The information in the background technology is only for illustrating the general background of the present invention and should not be regarded as admitting or suggesting in any form that such information constitutes the prior art known to a person skilled in the art. Summary of the invention

[0005] The present invention is based on the concept of DNA movable type storage and provides a method, a connection adapter library and a kit for constructing a DNA movable type storage unit. The construction method of the present invention mainly includes the following key steps: generation of a connection adapter library and combination screening thereof, generation of a DNA movable type library and screening thereof, design of a DNA movable type storage vector, and connection of key elements of a DNA movable type storage unit. Specifically, the present invention includes the following contents.

[0006] In a first aspect of the present invention, there is provided a method for constructing a DNA movable type storage unit, comprising the following steps:

[0007] Design and screen a connection adapter library;

[0008] Providing a DNA type library, the DNA type library comprising DNA type storing index, content and verification information;

[0009] Converting the digital information (such as 0-1 binary) of the computer target storage file into quaternary DNA base information, and retrieving a corresponding plurality of DNA active characters from the DNA active character library; and

[0010] The multiple movable characters are connected through necessary reactions, thereby completing the construction of the DNA movable character storage unit set and realizing the storage of the target file.

[0011] The method according to the first aspect of the present invention includes the following key steps: generation of a ligation linker library and its combination screening, generation of a DNA movable type library and its screening, design of a DNA movable type storage vector, and connection of key elements of a DNA movable type storage unit.

[0012] According to the method described in the first aspect of the present invention, the ligation adapter library comprises a plurality of ligation adapters capable of directional ligation, and the ligation adapters are sticky or blunt-ended nucleic acid sequences at the 5' or 3' end of the double-stranded nucleotide sequence of the DNA type.

[0013] According to the method described in the first aspect of the present invention, each of the ligands in the ligand library is composed of at least one of the four bases A, T, C, and G, and has a length of at least 1 base.

[0014] According to the method described in the first aspect of the present invention, the screening principles of the ligation adapter library include the number of consecutive bases being less than 3, the GC content being 20-80%, the number of base types being more than 1, and the number of self-dimers being less than 3 base pairs.

[0015] According to the method described in the first aspect of the present invention, the DNA characters are double-stranded nucleotide fragments formed by annealing single-stranded oligonucleotides.

[0016] According to the method described in the first aspect of the present invention, the DNA type mainly includes two parts: a linker sequence and a type body sequence, wherein the length of the type body sequence is customized and selected according to storage requirements, and the DNA type is divided into index type, content type and / or verification type according to its function, and the number of them is determined according to the design of the DNA type storage solution.

[0017] According to the method described in the first aspect of the present invention, the step of providing a DNA type library includes generating and screening DNA type, and the screening principles of the main sequence of the type during screening include a GC content of 20-80% and more than 3 base types.

[0018] According to the method described in the first aspect of the present invention, the step of generating a DNA type library mainly includes: based on the basic screening principle (GC content is 20-80%, the number of bases is 3 or more) of the main type sequence, the linker combination and the connection sites at both ends of the DNA type unit are preferentially selected according to the x-level directional connection requirement (x≥2, integer), the DNA type screening principle is customized, and the DNA type library is generated.

[0019] The method according to the first aspect of the present invention further comprises designing a DNA movable type storage carrier.

[0020] According to the method described in the first aspect of the present invention, the DNA movable type storage vector contains the necessary elements required for the live storage of engineered bacteria, such as DNA movable type unit insertion sites, resistance screening genes, and multiple cloning restriction sites; and can be obtained through chemical synthesis, storage plasmid enzyme digestion and recovery, and the like.

[0021] According to the method described in the first aspect of the present invention, the DNA movable type storage carrier is optimized to add at least one of an anchor sequence (adaptor), an index sequence and a sequencing primer sequence for high-throughput sequencing, thereby being used to construct a high-throughput sequencing self-built library sample in one step.

[0022] According to the method described in the first aspect of the present invention, the necessary reaction includes connecting movable type with different functions through one or more steps by biological enzyme preparations and / or chemical reagents to complete the construction of DNA movable type units, thereby realizing the storage of target files.

[0023] The second aspect of the present invention provides a ligation adapter library for constructing DNA type storage units, which includes a plurality of ligation adapters capable of directional ligation, wherein the ligation adapters can be ligated to the sticky / blunt end of the 5' or 3' end of the double-stranded nucleotide sequence of the DNA type.

[0024] The third aspect of the present invention provides a DNA type library, which comprises a plurality of double-stranded nucleotides as DNA types, each of which comprises a type main body sequence and an optional connecting linker sequence;

[0025] The main type sequence includes a sequence of specific digital information corresponding to the target file, and the connection linker sequence includes a sequence for connecting different storage types.

[0026] A fourth aspect of the present invention provides a kit for storing a target file, which comprises reagents for implementing the construction method according to the first aspect of the present invention.

[0027] According to the kit described in the fourth aspect of the present invention, the kit comprises at least one of a storage movable type, a storage carrier, a connection reagent and a buffer solution.

[0028] According to the kit described in the fourth aspect of the present invention, the ligation reagent of the kit comprises DNA ligase or other biological enzymes with similar functions, and the buffer solution comprises at least one chemical component selected from Tris-HCl, MgCl2, DTT, and ATP.

[0029] The present invention improves the efficiency of DNA type unit construction (i.e. data writing) by optimizing the combination of the connection adapter library of the DNA type unit. At the same time, the multi-fragment one-step directional connection combination of different levels provided by the present invention can be selected according to the requirements and costs (materials / labor) of different target files (or files to be stored).

[0030] The construction method of the DNA movable type storage unit of the present invention has the following advantages:

[0031] 1. Save costs and improve the efficiency of single-time information writing: The DNA movable type fragments required for the construction of DNA movable type storage units can be synthesized once and reused multiple times, saving costs;

[0032] 2. Flexible and diverse construction schemes: Researchers / users can select the number of DNA segment connection levels and match the optimal number of content / index / verification segments based on personalized needs such as the type, size, and storage cost of stored files. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The figure is a schematic diagram of the construction process of an exemplary DNA movable type storage unit of the present invention.

[0034] Figure 2 This is a schematic diagram of an exemplary DNA type classification of the present invention.

[0035] Figure 3 A schematic diagram of the screening regions generated for an exemplary DNA library of the present invention.

[0036] Figure 4 This is a schematic diagram of an exemplary DNA storage vector modification of the present invention.

[0037] Figure 5 A schematic diagram of an exemplary multi-stage connection of the present invention.

[0038] Figure 6 Agarose gel electrophoresis diagram showing PCR identification of ligation products of an exemplary DNA storage unit.

[0039] Figure 7 The transformation product of an exemplary DNA active type storage unit is blue-white (Amp + )Filter result graph.

[0040] Figure 8 Agarose gel electrophoresis diagram of PCR identification of ligation products of another exemplary DNA type storage unit.

[0041] Figure 9 The transformation product of another exemplary DNA active type storage unit is blue-white (Amp + )Filter result graph. DETAILED DESCRIPTION

[0042] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0043] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that the upper and lower limits of the scope and each intermediate value therebetween are specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0044] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0045] In this article, the term "ligation adapter library" refers to a large number of candidate adapters for DNA ligation, from which researchers / users can select the best adapter combination according to the required level of ligation. Generally speaking, the ligation adapter library is a virtual concept, not an independent physical library composed of multiple or multiple individual ligation adapters.

[0046] Herein, the term "DNA type" refers to a double-stranded nucleotide fragment or sequence of a specific digital information unit in a target file. The digital information unit in the target file can be repeated, so such double-stranded nucleotides can be used multiple times when the DNA is stored in the code, and are sometimes referred to as "type" or "storage type" herein. The corresponding digital information in the DNA type can correspond to information such as index, content or verification. The digital information unit can correspond to, for example, 0-1 binary, Chinese characters, letters, words, phrases, short phrases or punctuation marks, etc.

[0047] In this article, the term "DNA type library" refers to a combination of many independently existing DNA type libraries. Generally speaking, according to the function of DNA type, it can be divided into three types: index type library, content type library, and verification type library; according to the position of DNA type in the DNA type storage unit, it can be divided into two types: terminal type library and mid-end type library. Among them, the terminal type refers to the type located at the end of the constructed DNA type storage unit. Unlike the mid-end type, one end of the terminal type is connected to other structures such as a carrier, and only one end is connected to the mid-end type, while the two ends of the mid-end type are respectively connected to different types.

[0048] Herein, the term "DNA type storage unit" refers to a storage unit carrying certain digital information obtained by connecting or combining multiple DNA types selected from a DNA type library, which corresponds to a connected fragment containing multiple DNA type sequences.

[0049] The first aspect of the present invention provides a method for constructing a DNA movable type storage unit, which is sometimes referred to herein as "the construction method of the present invention", and is based on the idea of ​​DNA movable type storage. First, a connection joint library (preferably a multi-level connection joint library) is designed and screened, and the required joint combination is preferentially screened from the library according to the connection level; secondly, a DNA movable type library is provided, and the DNA movable type library contains DNA movable type for storing index, content and verification information. When storing data, the digital information (such as characters, pixels, audio amplitude, etc.) of the file to be stored (text, picture, audio, video, etc.) is converted into quaternary DNA base information according to relevant principles, and according to the DNA movable type storage scheme of the target file, a corresponding plurality of DNA movable types are selected or retrieved from the DNA movable type physical library (the physical library mainly includes content movable type, index movable type and verification movable type), and multi-level connection of the DNA movable type storage unit is performed to realize data information writing (see Appendix). Figure 1 ).

[0050] The construction of the DNA movable type storage unit involved in the present invention is mainly based on the DNA movable type storage unit construction plan of the file to be stored, and x DNA movable types (content / index / verification movable types) in the DNA movable type physical library are sequentially and directionally connected (x≥2) through a connection reaction such as an enzyme connection reaction in one or more steps to form a DNA movable type unit set composed of a large number of DNA movable type storage units.

[0051] The key steps of constructing the DNA movable type storage unit of the present invention mainly include:

[0052] (1) Screening of ligation adapter libraries and their combinations;

[0053] (2) DNA type library generation and physical library construction;

[0054] (3) Design of DNA movable type storage vector; and

[0055] (4) Multi-level connection of key elements of DNA movable type storage unit.

[0056] Step (1):

[0057] Step (1) of the present invention is preferably a multi-stage ligation adapter library and its combination screening, which mainly includes the following:

[0058] 1. Complete x-level one-step directional connection in the form of sticky / blunt ends (joints) at the 5' end of the nucleotide sequence corresponding to the DNA type, where x ≥ 2 (integer), such as 4 levels, 6 levels, 10 levels, 15 levels, 20 levels, 30 levels, etc.

[0059] 2. The sticky end adapter consists of n bases (n≥1), and the base type is at least one of the four types of A / C / G / T.

[0060] 3. Screening of adapter combinations, the screening method is not limited. For example, based on the adapter library screening principle shown in Table 1 below, the adapter combination screening principle is customized according to the x-level directional connection requirements (x≥2) and the personalized selection of the DNA storage vector.

[0061] Table 1 Principles of connector library screening

[0062]

[0063] Step (2):

[0064] Step (2) of the present invention is the generation and screening of a DNA type library, which mainly includes the following features:

[0065] 1. DNA is a double-stranded nucleotide fragment formed by annealing single-stranded oligonucleotides;

[0066] 2. The DNA type sequence mainly consists of two parts: the linker sequence and the main type sequence. The length of the main type sequence can be customized according to storage requirements;

[0067] 3. DNA movable type is divided into index movable type, content movable type and verification movable type according to its function. Their number can be determined according to the design of DNA movable type storage plan;

[0068] 4. DNA movable type is divided into terminal movable type (located at the connection end of the DNA storage skeleton carrier) and middle movable type according to its position in the construction of the DNA movable type storage unit. Their number can be determined according to the design of the DNA movable type storage plan;

[0069] 5. The generation of the DNA type library can be based on the type main sequence screening principle shown in Table 2 below, and the linker combination (see step 1 for details) selected according to the x-level directional connection requirements (x≥2) and the connection sites at both ends of the DNA type unit, and the DNA type screening principle is customized to generate the DNA type library.

[0070] Table 2 Principles for selecting movable type main sequence

[0071]

[0072] It should be noted that the main sequence of the movable type mainly carries specific data information. In order to ensure the diversity of the information, the length is set to be more than 10bp, preferably more than 15bp, and more preferably more than 20bp, taking into account the characteristics of the enzyme-linked reaction and the synthesis cost. On the other hand, the base types in the main sequence of the movable type are more than 3, such as 4. The GC content is 20-80%, preferably 30-60%, such as 40%, 50%. Combined with the aforementioned joint combination screening and the connection sites at both ends of the DNA movable type unit, in the generation of the DNA movable type sequence, the movable type main sequence and the joint sequence are combined for further sequence cleaning and sub-library division, and the "dirty" sequences with characteristics such as continuous repeated bases (≥3nt), too high GC content (>60%) or too low (<40%) are filtered, and the edit distance of the movable type sequence in the library is set to ≥8, reducing the sequence similarity in the library and improving the decoding success rate. After screening, the complete oligonucleotide sequence of the movable type in the library is finally obtained, and the forward and reverse chain sequences of different movable types are independently stored after synthetic annealing treatment to form a DNA movable type physical library.

[0073] In the actual storage process, the active type in the DNA active type physical library can be repeatedly called up to participate in the construction of different DNA active type units, thereby reducing the cost and errors caused by de novo synthesis of DNA sequences.

[0074] Step (3):

[0075] Step (3) of the present invention is the design and optimization of the DNA movable type storage carrier, which mainly includes the following features:

[0076] 1. The vector contains the necessary elements required for cloning and screening, which are obtained by enzyme digestion recovery / chemical synthesis / PCR amplification and are used for the construction of DNA movable type storage units. The necessary elements are not limited, and examples thereof include but are not limited to enzyme cutting sites, resistance screening genes, etc.

[0077] 2. The vector is preferably modified and optimized. For example, one of the anchor sequences (adaptor), index sequence or sequencing primer sequence for high-throughput sequencing is introduced at both ends of the connection site. Optionally, two or more combinations are introduced into the vector, and after the construction of the DNA movable type storage unit is completed, it is used to prepare self-built library sequencing samples, saving sequencing costs and shortening sequencing time.

[0078] Step (4):

[0079] Step (4) of the present invention is the construction of a DNA movable type storage unit, which mainly includes the following features:

[0080] 1. Use reagents (including biological enzyme preparations or chemical reagents or a combination thereof) to connect and construct DNA movable type carrying relevant target file storage information through one or more steps of enzyme connection or chemical reaction.

[0081] 2. The constructed DNA movable type storage unit preferably contains file information such as index / content / verification of the target storage file.

[0082] 3. The DNA movable type storage unit is preferably constructed by a one-step enzymatic reaction, but the present invention is not limited to a one-step enzymatic reaction, and the DNA movable type storage unit can also be constructed by any known method.

[0083] 4. Exemplarily, the enzymatic reaction system includes, in addition to the aforementioned DNA active type and storage carrier, other reagents required for the reaction, such other reagents are known in the art, examples of which include but are not limited to Tris-HCl, MgCl2, DTT, ATP, buffer ions, etc. The present invention may include one or more of such reagents.

[0084] The construction method of the invention can realize the directional connection of different DNA active words at one time to obtain the required DNA active word storage unit.

[0085] In a second aspect of the present invention, a ligation adaptor library for constructing a DNA movable type storage unit is provided, which comprises a plurality of ligation adaptors capable of directional connection, wherein the ligation adaptors can be connected to the sticky / blunt end at the 5' or 3' end of the double-stranded nucleotide sequence of the DNA movable type. The ligation adaptor library and its construction are not specifically limited, and the details can be found in the description of step (1) in the first aspect of the present invention, which will not be repeated here.

[0086] According to a third aspect of the present invention, a DNA type library is provided, which comprises a plurality of double-stranded nucleotide fragments (sometimes referred to herein as "double-stranded nucleotide sequences", the two being used interchangeably) as DNA types, each of which comprises a type body sequence and an optional linker sequence; wherein the type body sequence comprises specific digital information corresponding to a target file, and the linker sequence is used for connecting different stored types and also plays a role in positioning the types.

[0087] In certain embodiments, the DNA type library of the present invention comprises a plurality of type sub-libraries, and the joint sequence of each DNA type in each sub-library is the same but the main sequence of the type is different. The joint sequence is designed so that each type sub-library in the same DNA type library is docked with only one type sub-library in one direction, and no connection occurs between different types in the same group. For example, in the case where the DNA type library comprises the first type sub-library, the second type sub-library, the third type sub-library and the fourth type sub-library, any type in the first type sub-library is only directionally connected to one end of any type in the second type sub-library, but cannot be connected to the other end of the type, nor can it be connected to any type in the third type sub-library or the fourth type sub-library. The number y of type sub-libraries is not limited. Generally speaking, the number y of type sub-libraries is 2 or more, such as more than 4, more than 6, more than 10, more than 15, more than 20, more than 30, more than 50, more than 80, more than 100, etc. The number of types in each type sub-library is not limited.

[0088] In an exemplary embodiment, in order to achieve directional connection of DNA characters, the adapter sequences at the connecting ends of the first character sub-library and the second character sub-library are reverse complementary, and are not complementary to the connecting adapters in other sub-libraries in the DNA character library, nor do they form dimers.

[0089] In certain embodiments, the DNA active word library of the present invention comprises a first terminal active word sub-library and a second terminal active word sub-library, wherein the linker at one end of each active word in the first terminal active word sub-library or the second terminal active word sub-library is connected to a vector instead of being connected to other active words.

[0090] In a fourth aspect of the present invention, a kit for storing a target file is provided, which is based on DNA movable type storage technology and is used to store the target file in the form of DNA. The present invention is sometimes referred to as the "kit of the present invention", which comprises a storage movable type, a storage carrier, a connection reagent and a buffer solution. The storage movable type, i.e., the DNA movable type, has been described above and will not be repeated here.

[0091] In the present invention, the storage vector is any type of vector or plasmid that can insert a specific sequence for self-replication. The present invention can use known vectors, and can also design a dedicated vector as needed.

[0092] In the present invention, the ligation reagent is not limited, and includes various types of ligases, such as T4 DNA ligase, etc. The ligation reagent may also include chemical reagents for ligation, etc.

[0093] In the present invention, the buffer solution is not limited and can be any buffer solution suitable for DNA ligation. Exemplarily, the buffer solution includes chemical components such as Tris-HCl, MgCl2, DTT, ATP, etc. These chemical components can exist alone, in combination or in a mixed form.

[0094] Example

[0095] The following is an exemplary embodiment of the present invention. For the purpose of convenience of explanation, a movable type main body sequence length of 20 bp is used as an example, but the present invention is not limited thereto.

[0096] Embodiment 1

[0097] This example is an exemplary multi-stage ligation adapter library and its combination screening example.

[0098] In the construction of DNA movable type storage unit, the selection of linker combination is customized according to the length of linker, DNA storage vector and the selection of connection site. The DNA storage vector selected in this embodiment is LC-free (see Example 3 for details), the linker length is 4-base, and according to the screening principle of the aforementioned Table 1, a 4-base linker library with a storage capacity of 200 can be obtained (see Table 3 for details), and according to the linker combination screening scoring principle of Table 4 below, linker combinations of different directional connection levels are selected preferentially.

[0099] Table 3 4-base connector library

[0100] Serial number Connector Serial number Connector Serial number Connector Serial number Connector Serial number Connector 1 AATG 41 ACTA 81 TGGA 121 GTGA 161 CACA 2 AATC 42 ACTT 82 TGGT 122 GTGT 162 CACT 3 AAGA 43 ACTG 83 TGGC 123 GTGG 163 CACG 4 AAGT 44 ACTC 84 TGCT 124 GTGC 164 CACC 5 AAGG 45 ACGA 85 TGCG 125 GTCA 165 CTAA 6 AAGC 46 ACGG 86 TGCC 126 GTCT 166 CTAT 7 AACA 47 ACGC 87 TCAA 127 GTCG 167 CTAC 8 AACT 48 ACCA 88 TCAT 128 GTCC 168 CTTA 9 AACG 49 ACCT 89 TCAG 129 GGAA 169 CTTG 10 AACC 50 ACCG 90 TCAC 130 GGAT 170 CTTC 11 ATAG 51 TAAG 91 TCTA 131 GGAG 171 CTGA 12 ATAC 52 TAAC 92 TCTT 132 GGAC 172 CTGT 13 ATTG 53 TATG 93 TCTG 133 GGTA 173 CTGG 14 ATTC 54 TATC 94 TCTC 134 GGTT 174 CTGC 15 ATGA 55 TAGA 95 TCGT 135 GGTG 175 CTCA 16 ATGT 56 TAGT 96 TCGG 136 GGTC 176 CTCT 17 ATGG 57 TAGG 97 TCGC 137 GGCA 177 CTCG 18 ATGC 58 TAGC 98 TCCA 138 GGCT 178 CTCC 19 ATCA 59 TACA 99 TCCT 139 GCAA 179 CGAA 20 ATCT 60 TACT 100 TCCG 140 GCAT 180 CGAT 21 ATCG 61 TACG 101 GAAT 141 GCAG 181 CGAG 22 ATCC 62 TACC 102 GAAG 142 GCAC 182 CGAC 23 AGAA 63 TTAG 103 GAAC 143 GCTA 183 CGTA 24 AGAT 64 TTAC 104 GATA 144 GCTT 184 CGTT 25 AGAG 65 TTGA 105 GATT 145 GCTG 185 CGTG 26 AGAC 66 TTGT 106 GATG 146 GCTC 186 CGTC 27 AGTA 67 TTGG 107 GAGA 147 GCGA 187 CGGA 28 AGTT 68 TTGC 108 GAGT 148 GCGT 188 CGGT 29 AGTG 69 TTCA 109 GAGG 149 GCCA 189 CGCA 30 AGTC 70 TTCT 110 GAGC 150 GCCT 190 CGCT 31 AGGA 71 TTCG 111 GACA 151 CAAT 191 CCAA 32 AGGT 72 TTCC 112 GACT 152 CAAG 192 CCAT 33 AGGC 73 TGAA 113 GACG 153 CAAC 193 CCAG 34 AGCA 74 TGAT 114 GACC 154 CATA 194 CCAC 35 AGCG 75 TGAG 115 GTAA 155 CATT 195 CCTA 36 AGCC 76 TGAC 116 GTAT 156 CATC 196 CCTT 37 ACAA 77 TGTA 117 GTAG 157 CAGA 197 CCTG 38 ACAT 78 TGTT 118 GTTA 158 CAGT 198 CCTC 39 ACAG 79 TGTG 119 GTTG 159 CAGG 199 CCGA 40 ACAC 80 TGTC 120 GTTC 160 CAGC 200 CCGT

[0101] Table 4 Scoring principles for 4-base connector combination screening

[0102]

[0103] Note: The original score of the connector is 100. The corresponding score will be deducted based on the above situation, and the connector combination will be selected based on the actual storage needs.

[0104] Embodiment 2

[0105] This example is an example of the generation and screening of an exemplary DNA character library.

[0106] The DNA type in this embodiment is a double-stranded nucleotide fragment formed by annealing a single-stranded oligonucleotide. The DNA type sequence includes two parts: a linker and a main type sequence. According to the functional category represented by the DNA type, it can be divided into index type, content type, and verification type, and their number can be determined according to the design of the DNA type storage scheme. According to the position of the DNA type in the construction of the DNA type storage unit, it can be divided into terminal type (located at the connection end of the DNA storage backbone vector) and mid-terminal type. Taking the DNA storage vector LC-free (see Example 3 for details), the terminal connection site HindIII / EcoRI, and 10-level connection as an example, the type classification schematic diagram is shown in the attached Figure 2 .

[0107] The length of the main type sequence used in this embodiment is 20 bp, and the DNA storage vector is LC-free. First, according to the above main type sequence screening principle (see Table 2 for details), the main type sequence library is obtained. On this basis, for different screening regions (see Appendix for details), Figure 3 ), generate the required movable type library according to the movable type screening principles in Table 5 below.

[0108] Table 5 DNA type screening principles

[0109]

[0110] Embodiment 3

[0111] This embodiment is an example of optimization of an exemplary DNA movable type storage carrier.

[0112] The DNA movable type storage vector used in this embodiment is pUC19, which is used as the original vector for transformation and optimization. The transformed storage vector is named: Library construction-free plasmid (abbreviated as: LC-free) (see the schematic diagram for details) Figure 4 ).

[0113] The specific steps are as follows: 1) using pUC19 plasmid as a template, single restriction digestion with HindIII was performed, and a double-stranded nucleotide fragment after HF / HR annealing was connected, which contained a P5 anchor sequence + P5 index + Read1 sequencing primer; 2) after transformation, single clone selection, and PCR identification (identification primer pair is 524F / 881R), a positive clone was obtained and named H19; 3) using H19 plasmid as a template, single restriction digestion with EcoRI was performed, and a double-stranded nucleotide fragment after EF / ER annealing was connected, which contained a P7 anchor sequence + P7index + Read2 sequencing primer; 4) after transformation, single clone selection, and PCR identification (identification primer pair is 524F / 881R), the positive clone obtained was the transformed storage vector, named Library construction-free plasmid.

[0114] Table 6 Related primer sequences

[0115]

[0116] Note: Underline indicates anchor sequence; bold indicates index sequence; dotted line indicates Read sequencing primer.

[0117] Embodiment 4

[0118] In this example, 4 to 12 levels of connection with a linker length of 4-base were tested. The DNA movable type storage vector used was LC-free (see Example 3 for details), and the movable type main body sequence length was 20 bp.

[0119] The specific test steps are as follows:

[0120] 1) According to the connector and combination screening principle of the above-mentioned embodiment 1, the connector required for the test is obtained (see Table 7 for details);

[0121] 2) According to the principles of type generation and screening in the second embodiment, a relevant type library is obtained (see Tables 8 and 9 for details);

[0122] 3) The single-stranded oligonucleotide sequence of the active type is synthesized and annealed to obtain the active type fragment (insert) required for the test experiment;

[0123] 4) Using the LC-free plasmid as a template, double digestion with HindIII / EcoRI was performed to recover the ligation vector;

[0124] 5) According to the relevant connection system, complete the 4th to 12th level connection in 9 centrifuge tubes respectively, and carry out: a. PCR amplification and sequencing of the connection products (see the attached agarose gel for details) Figure 6 : M is a marker, indicating 100bp, 250bp, 500bp, 750bp, 1000bp, and 2000bp from bottom to top; CN is an empty control of 514bp; 4 to 12 are PCR amplification fragments of the corresponding connection levels, with sizes of 561bp, 585bp, 609bp, 633bp, 657bp, 681bp, 705bp, 729bp, and 753bp, respectively; b. The blue-white color of the transformation product (Amp + ) Clone identification (see attached for details of the plate map) Figure 7 ).

[0125] It has been verified that the PCR amplification sequencing results are correct and the positive cloning rate is high. The results show that the method of this embodiment is effective and can be used to construct a DNA movable type storage unit, and provides an efficient and variable storage solution for DNA movable type storage.

[0126] Table 7 Multi-level connection test joint table (4-base)

[0127]

[0128] Table 8 Multi-level connection test type table (4-base)

[0129]

[0130]

[0131] Table 9 Multi-level connection test character sequence list (4-base)

[0132]

[0133]

[0134] Embodiment 5

[0135] This example tests the 4 to 12 level connections with a linker length of 6 bases, the DNA type storage carrier used is LC-free (see Example 3 for details), and the length of the main type sequence is 20 bp. The specific test steps are as follows:

[0136] 1) According to the relevant principles, the connectors (see Table 10 for details) and movable type (see Tables 11 and 12 for details) required for the test are screened / generated respectively;

[0137] 2) The single-stranded oligonucleotide sequence of the active type is synthesized and annealed to obtain the active type fragment (insert) required for the test experiment;

[0138] 3) Using the LC-free plasmid as a template, double digestion with HindIII / EcoRI was performed to recover the ligation vector;

[0139] 4) According to the relevant connection system, complete the 4th to 12th level connection in 9 centrifuge tubes respectively, and carry out: a. PCR amplification and sequencing of the connection products (see the attached agarose gel for details) Figure 8 : M is a marker, indicating 100bp, 250bp, 500bp, 750bp, 1000bp, and 2000bp from bottom to top; CN is an empty control of 514bp; 4 to 12 are PCR amplification fragments of the corresponding connection levels, with sizes of 567bp, 593bp, 619bp, 645bp, 671bp, 697bp, 723bp, 749bp, and 775bp, respectively); b. Identification of blue-white (Amp+) clones of the transformation products (see the attached plate map for details). Figure 9 ).

[0140] Table 10 Multi-level connection test joint table (6-base)

[0141]

[0142] Table 11 Multi-level connection test type table (6-base)

[0143]

[0144]

[0145] Table 12 Multi-level connection test type sequence table (6-base)

[0146]

[0147]

[0148] It has been verified that the PCR amplification sequencing results are correct and the positive cloning rate is high. The results show that the method of this embodiment is effective and can be used to construct a DNA movable type storage unit, and provides an efficient and variable storage solution for DNA movable type storage.

Claims

1. A method for constructing a DNA movable type storage unit, characterized in that: The following steps are involved: Design and screen a connection adapter library; Providing a DNA type library, the DNA type library comprising DNA type storing index, content and verification information; Converting the digital information of the computer target storage file into quaternary DNA base information, and retrieving a corresponding plurality of DNA movable characters from the DNA movable character library; and The multiple movable types are connected through necessary reactions to complete the construction of the corresponding DNA movable type storage unit, and the multiple necessary DNA movable type storage units constitute a DNA movable type storage unit set, thereby achieving the purpose of DNA movable type storage of the target file.

2. The method according to claim 1, characterized in that The method comprises the following key steps: generation of a connection adapter library and combination screening thereof, generation of a DNA movable type library and screening thereof, design of a DNA movable type storage vector, and connection of key elements of a DNA movable type storage unit.

3. The method according to claim 1 or 2, characterized in that: The ligation adapter library comprises a plurality of ligation adapters capable of directional ligation, wherein the ligation adapters are nucleic acid sequences with sticky or blunt ends at the 5' or 3' ends of the double-stranded nucleotide sequence of the DNA type.

4. The method according to any one of claims 1 to 3, characterized in that: The ligation adapters in the ligation adapter library are each composed of at least one of the four bases A, T, C, and G, and their length includes at least 1 base.

5. The method according to any one of claims 1 to 4, characterized in that: The screening principles of the ligation adapter library include less than 3 consecutive bases of the same type, 20-80% GC content, more than 1 base type, and less than 3 base pairs of self-dimers.

6. The method according to claim 1 or 2, characterized in that: DNA is a double-stranded nucleotide fragment formed by annealing single-stranded oligonucleotides.

7. The method according to any one of claims 1, 2 or 6, characterized in that: The DNA type mainly includes two parts: a linker sequence and a main type sequence. The length of the main type sequence is customized according to storage requirements, and the DNA type is divided into index type, content type and / or verification type according to its function, and the number of them is determined according to the design of the DNA type storage solution.

8. According to the method described in any one of claims 1, 2, 6 or 7, the step of providing a DNA type library includes generating and screening DNA type, and the screening principles of the main sequence of the type during screening include a GC content of 20-80% and more than 3 base types.

9. According to the method described in any one of claims 1, 2, 6-8, the step of generating the DNA active type library mainly comprises: Based on the basic screening principle (GC content of 20-80%, base types of 3 or more) of the main sequence of the type, the linker combination and the connection sites at both ends of the DNA type unit are preferentially selected according to the x-level directional connection requirements (x≥2, integer), and the DNA type screening principle is customized to generate a DNA type library.

10. The method according to claim 1 or 2, characterized in that: It further includes designing a DNA movable type storage carrier.

11. The method according to claim 1, 2, 10, characterized in that: The DNA active type storage vector contains the necessary elements required for the live storage of engineered bacteria, such as DNA active type unit insertion sites, resistance screening genes, and multiple cloning restriction sites; and can be obtained through chemical synthesis, storage plasmid enzyme digestion and recovery, and other methods.

12. The method according to claim 1, 2, 10 or 11, characterized in that The DNA movable type storage carrier is optimized in design to add at least one of an anchor sequence, an index sequence and a sequencing primer sequence for high-throughput sequencing, so as to be used for constructing a high-throughput sequencing self-built library sample.

13. The method according to claim 1 or 2, characterized in that: The necessary reaction includes connecting movable characters with different functions through one or more steps by biological enzyme preparations and / or chemical reagents to complete the construction of DNA movable character units, thereby realizing the storage of target files.

14. A connection adapter library for constructing a DNA movable type storage unit, characterized in that: Includes a variety of ligation adapters capable of directional ligation, which can be ligated to the sticky / blunt ends of the 5' or 3' ends of the double-stranded nucleotide sequence of the DNA type.

15. A DNA movable type library, characterized in that: It comprises a plurality of double-stranded nucleotides as DNA characters, each of which comprises a character main body sequence and an optional connecting linker sequence; The main type sequence includes a sequence of specific digital information corresponding to the target file, and the connection linker sequence includes a sequence for connecting different storage types.

16. A kit for storing a target file, characterized in that: The invention comprises reagents for implementing the construction method according to any one of claims 1 to 13.

17. The kit according to claim 16, characterized in that The kit comprises at least one of a storage movable type, a storage carrier, a connection reagent and a buffer solution.

18. The kit according to claim 16 or 17, characterized in that: The ligation reagent of the kit comprises DNA ligase or other biological enzymes with similar functions, and the buffer solution comprises at least one chemical component of Tris-HCl, MgCl2, DTT, and ATP.

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