Use of nucleic acid molecule variants and methods of synthesis

Abstract

The application relates to the fields of biotechnology and information storage technology, and particularly relates to an application and a synthesis method of a nucleic acid molecule variant; the nucleic acid molecule variant is composed of nucleotide homopolymers; the nucleotide homopolymers include homodimers, homotrimers, homotetramers, homopentamers, homohexamers, homoheptamers and homo-octamers; when used as a storage medium, the nucleic acid molecule variant takes the nucleotide homopolymers or conversion thereof as a basic unit for information coding, and high-fidelity data storage can be realized; a suitable polymerase variant and a template-dependent enzymatic synthesis method are developed for synthesizing the nucleic acid molecule variant.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and information storage technology, specifically to the application and synthesis method of a nucleic acid molecular variant. Background Technology

[0002] Data storage is one of the most important potential applications of DNA. Compared to traditional hard drives, magnetic tapes, or optical discs, DNA storage offers disruptive advantages such as ultra-high density, ultra-long lifespan, and low energy consumption, making it a powerful solution for massive data storage in the future. During DNA synthesis, storage, and sequencing, nucleotide deletions, insertions, or substitutions are easily caused, making accurate information retrieval difficult. While many encoding methods can improve information redundancy and thus accuracy, designing nucleic acid molecules to improve information accuracy from the ground up has a simple yet powerful advantage.

[0003] There are two main methods for synthesizing nucleic acid molecules: chemical methods and enzymatic methods. Currently, chemical synthesis, which relies on phosphorus amide, is dominant. However, because the coupling efficiency is at most around 99.5%, chemical synthesis is unlikely to produce DNA with more than 300 nucleotides. Furthermore, chemical synthesis requires the use of large amounts of toxic and harmful organic solvents, such as acetonitrile, which also burdens the environment.

[0004] Enzymatic synthesis, requiring mild reaction conditions, is highly efficient, low-cost, and environmentally friendly, making it an ideal alternative to chemical synthesis. Currently, enzymatic synthesis primarily relies on terminal deoxynucleotidyl transferase (TdT), a polymerase that can synthesize DNA de novo without a template. Based on TdT, researchers have developed methods such as TdT-dNTP covalent coupling, reversible terminator (RT-dNTP) methods, TdT-apyrase coupling, and photocontrolled methods to achieve controllable enzymatic synthesis of DNA. However, compared to chemical methods, the synthesis efficiency of enzymatic methods remains relatively low. Moreover, in TdT-based enzymatic synthesis, as the DNA chain lengthens, single-stranded DNA may form secondary structures, further affecting synthesis efficiency. The purity and price of TdT-dNTP conjugates and RT-dNTPs are also limiting factors for their correct synthesis and commercial application.

[0005] To address these issues, this invention proposes using nucleic acid molecule variants as storage media, employing homopolymers of nucleotides instead of individual nucleotides as the basic unit for information encoding for data storage; and develops suitable polymerase variants to construct a template-dependent enzymatic synthesis method for synthesizing these nucleic acid molecules. Using this polymerase for information encoding and decoding, high-fidelity data storage using this nucleic acid molecule variant as the medium is achieved. Summary of the Invention

[0006] The purpose of this invention is to provide an application and synthesis method for nucleic acid molecular variants, so as to solve the problems of DNA data storage and enzymatic synthesis in the background art.

[0007] This invention utilizes homodimers, homotrimers, homotetramers, homopentamers, homohexamers, homoheptamers, and homooctamers of nucleotides to replace single nucleotides as the basic unit for information encoding in data storage.

[0008] When DNA is used as a medium for information storage, information is typically represented by a single nucleotide or a transformation of nucleotides. However, errors can occur during synthesis and reading; the deletion, insertion, or mutation of a single nucleotide can lead to deviations in the encoded information, requiring complex error correction procedures. This invention uses nucleotide homopolymers instead of single nucleotides as the basic unit of information encoding. Even if a nucleotide mutates, the correct nucleotide can be deduced based on the nucleotide homopolymer encoding principle and neighboring nucleotides, thereby simply and conveniently improving the accuracy of information storage.

[0009] This invention yields a series of polymerase variants that can efficiently and accurately achieve de novo synthesis of the aforementioned nucleic acid molecules under the guidance of a given template.

[0010] Template-dependent nucleic acid molecule synthesis typically requires that the primer and template have at least 18 complementary bases, meaning the template must contain at least 18 nucleotides. If an additional nucleotide needs to be added to the primer, a template containing at least 19 nucleotides is required. To synthesize any nucleic acid molecule, 4... 19 There are several templates required. However, if a nucleic acid molecule variant composed of homopolymers is synthesized, the number of templates required is significantly reduced. For example, to synthesize a homotrimeric nucleic acid molecule, if the primer and template are 18 bases complementary, and one homotrimer is added, meaning the core region of the template has 21 nucleotides, the required number of templates is 4 × 3. 21 / 3-1 =2916 kinds, far less than 4 19 The polymerase variant obtained in this invention enables precise synthesis of nucleic acid molecule variants even when the primer and template are only 9 bases complementary; that is, when the core region of the template contains only 12 nucleotides, three nucleotides can be accurately added to the 3' end of the primer. This allows for the precise addition of three nucleotides to the 3' end of the primer with only 4 × 3... 12 / 3-1 With 108 templates, any homotrimeric nucleic acid molecule variant can be synthesized.

[0011] This invention constructs a template-dependent enzymatic synthesis method to synthesize this nucleic acid molecule variant, and uses it for information encoding and decoding. This method uses natural, unmodified nucleotide substrates, achieves a nucleic acid molecule synthesis accuracy of 99.51%, and enables lossless encoding and decoding of information.

[0012] To achieve the above objectives, the present invention provides the following technical solution:

[0013] This invention provides a method for synthesizing nucleic acid molecule variants. The method involves designing and synthesizing starter primers and a template; immobilizing the primers on a solid support; adding the template, polymerase variant, and target nucleotide molecules to perform an enzymatic reaction, adding a given number of target nucleotide molecules to the 3'-terminus of the primers. Then, the above steps are repeated sequentially according to the target nucleic acid molecule sequence, adding the corresponding nucleotides in sequence. After each addition, the template, polymerase variant, and unreacted nucleotide molecules are washed with a washing solution. After the nucleic acid molecule synthesis is complete, it is peeled off from the solid support to obtain the synthetic product. The nucleic acid molecule variant is composed of nucleotide homopolymers; the nucleotide homopolymers include homodimers, homotrimers, homotetramers, homopentamers, homohexamers, homoheptamers, homooctamers, etc.

[0014] In some implementations, the polymerase variants include Taq DNA polymerase variants and Dpo4 polymerase variants.

[0015] In some embodiments, the Taq DNA polymerase variant includes: TaqΔN-TM, the amino acid sequence of which is shown in SEQ ID No. 1; the Dpo4 polymerase variant includes Dpo4ΔC and Dpo4ΔC-CL7, the amino acid sequence of which is shown in SEQ ID No. 2, and the amino acid sequence of which is shown in SEQ ID No. 3.

[0016] The synthesis method specifically includes the following steps:

[0017] (1) Design and synthesize the starting primer (N);

[0018] (2) Fix the starting primer (N) obtained in step (1) onto a solid support;

[0019] (3) Add reaction buffer, target nucleotide molecule, template and any one of the polymerase variants mentioned above to the product obtained in step (2);

[0020] (4) Denaturation-annealing-extension: Add a given number of target nucleotide molecules to the 3' end of the primer. The specific number depends on the type of homopolymer. For example, for homotrimer, add three target nucleotide molecules to the 3' end of the primer.

[0021] (5) Wash to remove template, polymerase variant and unreacted target nucleotide molecules, etc., to obtain primers with 1 homopolymer added (N+1).

[0022] (6) Repeat steps (3)-(5) until the synthesis of the given sequence (nucleic acid molecular variant sequence) is complete;

[0023] (7) The product is peeled off from the solid support to obtain a nucleic acid molecular variant.

[0024] In some implementations, in step (3), the template is a single-stranded nucleic acid molecule variant, which consists of a core region and protective base portions at the 5' and 3' ends on both sides; the core region consists of two parts: a primer complementary portion and a guiding base portion; the primer complementary portion is complementary to the 3' end base of the primer; the guiding base portion guides the addition of nucleotides complementary to its bases; the primer complementary portion and the guiding base portion are each composed of nucleotide homopolymers.

[0025] In some implementations, the 3' protective base portion consists of at least three nucleotides, which can effectively enhance the efficiency of nucleic acid molecule synthesis. For the 5' protective base portion, the last nucleotide differs from the first nucleotide in the template core region.

[0026] In some implementations, the 5' end protective base moiety may not be required.

[0027] In step (3), the total number of templates, Total, is obtained by the following formula:

[0028]

[0029] Where L is the number of nucleotides in the template core region, generally not exceeding 21 nucleotides; n is the number of nucleotides in a single homopolymer; the value of L / n is a positive integer. For example, if the homopolymer is a homotrimer and the number of nucleotides in the template core region is 21, then the total number is 4 × 3. 21 / 3-1 =2916.

[0030] In some embodiments, the template library of the present invention comprises a series of single-stranded oligonucleotides complementary to the primers. Taking the synthesis of a nucleic acid molecule variant composed of homotrimers (n=3) as an example, if the 3' complementary portion between the template and the primer has 9 nucleotides, i.e., 3 homotrimers; when the template is needed to guide the addition of another homotrimer, the number of nucleotides increases to 12, i.e., four homotrimers; that is, the number of nucleotides in the core region of the template is 12, i.e., L=12. For example, the primer is 5'-AAA. TTTGGGTTT -3', when a homotrimer 5'-CCC-3' needs to be added; the core region of the template is 5'-GGG. AAACCCAAA-3'. Where 5'-AAACCCAAA-3' is the complementary portion to the 3' end of the primer, and 5'-GGG-3' is the portion guiding the addition of the new homotrimer 5'-CCC-3'. To synthesize any homotrimer-based nucleic acid variant, the core region of the template library must cover a combination of homotrimer variants with a nucleotide count of 12, i.e., the size of the template library or the total number of templates must be 4 × 3. 12 / 3-1 =108. The template library contains all possible combinations of three homotrimers as complementary regions, plus one homotrimer that may be needed to guide addition. Adjacent homotrimers are distinct, otherwise, such as 5'-CCC CCC-3', they are homohexamers. When the protecting bases also consist of homopolymers of nucleotides, only a small amount of initial template is required, and subsequent templates can be synthesized by the method provided in this invention and other low-cost methods. When synthesizing homotrimeric nucleic acid molecular variants, if a template library with 15 nucleotides in the core region is used, the sequence of the core region is shown in SEQ ID No. 87-410; if a template library with 12 nucleotides in the core region is used, the sequence of the core region is shown in SEQ ID No. 411-518.

[0031] SEQ ID No. 87-410:

[0032] ‘AAATTTAAATTTAAA’, ‘AAATTTAAATTTCCC’, ‘AAATTTAAATTTGGG’,‘AAATTTAAACCCAAA’, ‘AAATTTAAACCCTTT’, ‘AAATTTAAACCCGGG’, ‘AAATTTAAAGGGAAA’,‘AAATTTAAAGGGTTT’, ‘AAATTTAAAGGGCCC’, ‘AAATTTCCCAAATTT’, ‘AAATTTCCCAAACCC’,‘AAATTTCCCAAAGGG’, ‘AAATTTCCCTTTAAA’, ‘AAATTTCCCTTTCCC’, ‘AAATTTCCCTTTGGG’,‘AAATTTCCCGGGAAA’, ‘AAATTTCCCGGGTTT’, ‘AAATTTCCCGGGCCC’, ‘AAATTTGGGAAATTT’,‘AAATTTGGGAAACCC’, ‘AAATTTGGGAAAGGG’, ‘AAATTTGGGTTTAAA’, ‘AAATTTGGGTTTCCC’,‘AAATTTGGGTTTGGG’, ‘AAATTTGGGCCCAAA’, ‘AAATTTGGGCCCTTT’, ‘AAATTTGGGCCCGGG’,‘AAACCCAAATTTAAA’, ‘AAACCCAAATTTCCC’, ‘AAACCCAAATTTGGG’, ‘AAACCCAAACCCAAA’,‘AAACCCAAACCCTTT’, ‘AAACCCAAACCCGGG’, ‘AAACCCAAAGGGAAA’, ‘AAACCCAAAGGGTTT’,‘AAACCCAAAGGGCCC’, ‘AAACCCTTTAAATTT’, ‘AAACCCTTTAAACCC’, ‘AAACCCTTTAAAGGG’,‘AAACCCTTTCCCAAA’, ‘AAACCCTTTCCCTTT’, ‘AAACCCTTTCCCGGG’, ‘AAACCCTTTGGGAAA’,‘AAACCCTTTGGGTTT’, ‘AAACCCTTTGGGCCC’, ‘AAACCCGGGAAATTT’, ‘AAACCCGGGAAACCC’,‘AAACCCGGGAAAGGG’, ‘AAACCCGGGTTTAAA’, ‘AAACCCGGGTTTCCC’, ‘AAACCCGGGTTTGGG’,‘AAACCCGGGCCCAAA’, ‘AAACCCGGGCCCTTT’,'AAACCCGGGGCCCGGG', 'AAAGGGAAATTTAAA','AAAGGGAAATTTCCC', 'AAAGGGAAATTTGGG', 'AAAGGGAACCCAAA', 'AAAGGGAAACCCTTTT','AAAGGGAAACCCGG', 'AAAGGGAAAGGGAA', 'AAAGGGAAAGGGTTTT', 'AAAGGGAAAGGGCCC','AAAGGGTTTAAATTT', 'AAAGGGTTTAAACCC', 'AAAGGGTTTAAAGGG', 'AAAGGGTTTCCCAAA','AAAGGGTTTCCCTTTT', 'AAAGGGTTTCCCGG', 'AAAGGGTTTGGGAA', 'AAAGGGTTTGGGTTTT', 'AAAGGGTTTGGGCC', 'AAAGGGCCCAAATTT', 'AAAGGGCCCAAACCC', 'AAAGGGCCCAAAGGG','AAAGGGCCCCTTTAAA', 'AAAGGGCCCCTTTTTTAAAA', 'AAAGGGCCCCTTTTTTGG', 'AAAGGGCCCCGGGAAA','AAAGGGCCCCGGGTTT', 'AAAGGGCCCCGGGGAA','AAAGGGCCCCGGGTTT', 'AAAGGGCCCCGGCCCC, 'TTTAAATTTAAATTT', 'TTTAAATTTAAACCC','TTTAAATTTAAAGGG', 'TTTAAATTTCCCCAAA', 'TTTAAATTTCCCTTT', 'TTTAAATTTCCCGGG','TTTAAATTTGGGGAAA', 'TTTAAATTTGGGTTT', 'TTTAAATTTGGGCCC', 'TTTAAACCCAAATTT', 'TTTAAACCCAAACCC', 'TTTAAATTTCCCGGGG', 'TTTAAACCCTTTCCC','TTTAAACCCTTTGGG', 'TTTAAACCCGGGAAA', 'TTTAAACCCGGGTTT', 'TTTAAACCCGGGCCC','TTTAAAGGGAAATTT', 'TTTAAAGGGAAACCC', 'TTTAAAGGGAAAGGG', 'TTTAAAGGGTTTAAA','TTTAAAGGGTTTCCC', 'TTTAAAGGGTTTGGG', 'TTTAAAGGGCCCAAA',‘TTTAAAGGGCCCTTT’,‘TTTAAAGGGCCCGGG’, ‘TTTCCCAAATTTAAA’, ‘TTTCCCAAATTTCCC’, ‘TTTCCCAAATTTGGG’,‘TTTCCCAAACCCAAA’, ‘TTTCCCAAACCCTTT’, ‘TTTCCCAAACCCGGG’, ‘TTTCCCAAAGGGAAA’,‘TTTCCCAAAGGGTTT’, ‘TTTCCCAAAGGGCCC’, ‘TTTCCCTTTAAATTT’, ‘TTTCCCTTTAAACCC’,‘TTTCCCTTTAAAGGG’, ‘TTTCCCTTTCCCAAA’, ‘TTTCCCTTTCCCTTT’, ‘TTTCCCTTTCCCGGG’,‘TTTCCCTTTGGGAAA’, ‘TTTCCCTTTGGGTTT’, ‘TTTCCCTTTGGGCCC’, ‘TTTCCCGGGAAATTT’,‘TTTCCCGGGAAACCC’, ‘TTTCCCGGGAAAGGG’, ‘TTTCCCGGGTTTAAA’, ‘TTTCCCGGGTTTCCC’,‘TTTCCCGGGTTTGGG’, ‘TTTCCCGGGCCCAAA’, ‘TTTCCCGGGCCCTTT’, ‘TTTCCCGGGCCCGGG’,‘TTTGGGAAATTTAAA’, ‘TTTGGGAAATTTCCC’, ‘TTTGGGAAATTTGGG’, ‘TTTGGGAAACCCAAA’,‘TTTGGGAAACCCTTT’, ‘TTTGGGAAACCCGGG’, ‘TTTGGGAAAGGGAAA’, ‘TTTGGGAAAGGGTTT’,‘TTTGGGAAAGGGCCC’, ‘TTTGGGTTTAAATTT’, ‘TTTGGGTTTAAACCC’, ‘TTTGGGTTTAAAGGG’,‘TTTGGGTTTCCCAAA’, ‘TTTGGGTTTCCCTTT’, ‘TTTGGGTTTCCCGGG’, ‘TTTGGGTTTGGGAAA’,‘TTTGGGTTTGGGTTT’, ‘TTTGGGTTTGGGCCC’, ‘TTTGGGCCCAAATTT’, ‘TTTGGGCCCAAACCC’,‘TTTGGGCCCAAAGGG’, ‘TTTGGGCCCTTTAAA’, ‘TTTGGGCCCTTTCCC’, ‘TTTGGGCCCTTTGGG’,‘TTTGGGCCCGGGAAA’, ‘TTTGGGCCCGGGTTT’, ‘TTTGGGCCCGGGCCC’, ‘CCCAAATTTAAATTT’,‘CCCAAATTTAAACCC’, ‘CCCAAATTTAAAGGG’, ‘CCCAAATTTCCCAAA’, ‘CCCAAATTTCCCTTT’,‘CCCAAATTTCCCGGG’, ‘CCCAAATTTGGGAAA’, ‘CCCAAATTTGGGTTT’, ‘CCCAAATTTGGGCCC’,‘CCCAAACCCAAATTT’, ‘CCCAAACCCAAACCC’, ‘CCCAAACCCAAAGGG’, ‘CCCAAACCCTTTAAA’,‘CCCAAACCCTTTCCC’, ‘CCCAAACCCTTTGGG’, ‘CCCAAACCCGGGAAA’, ‘CCCAAACCCGGGTTT’,‘CCCAAACCCGGGCCC’, ‘CCCAAAGGGAAATTT’, ‘CCCAAAGGGAAACCC’, ‘CCCAAAGGGAAAGGG’,‘CCCAAAGGGTTTAAA’, ‘CCCAAAGGGTTTCCC’, ‘CCCAAAGGGTTTGGG’, ‘CCCAAAGGGCCCAAA’,‘CCCAAAGGGCCCTTT’, ‘CCCAAAGGGCCCGGG’, ‘CCCTTTAAATTTAAA’, ‘CCCTTTAAATTTCCC’,‘CCCTTTAAATTTGGG’, ‘CCCTTTAAACCCAAA’, ‘CCCTTTAAACCCTTT’, ‘CCCTTTAAACCCGGG’,‘CCCTTTAAAGGGAAA’, ‘CCCTTTAAAGGGTTT’, ‘CCCTTTAAAGGGCCC’, ‘CCCTTTCCCAAATTT’,‘CCCTTTCCCAAACCC’, ‘CCCTTTCCCAAAGGG’, ‘CCCTTTCCCTTTAAA’, ‘CCCTTTCCCTTTCCC’,‘CCCTTTCCCTTTGGG’, ‘CCCTTTCCCGGGAAA’, ‘CCCTTTCCCGGGTTT’, ‘CCCTTTCCCGGGCCC’,‘CCCTTTGGGAAATTT’, ‘CCCTTTGGGAAACCC’, ‘CCCTTTGGGAAAGGG’, ‘CCCTTTGGGTTTAAA’,‘CCCTTTGGGTTTCCC’,‘CCCTTTGGGTTTGGG’, ‘CCCTTTGGGCCCAAA’, ‘CCCTTTGGGCCCTTT’,‘CCCTTTGGGCCCGGG’, ‘CCCGGGAAATTTAAA’, ‘CCCGGGAAATTTCCC’, ‘CCCGGGAAATTTGGG’,‘CCCGGGAAACCCAAA’, ‘CCCGGGAAACCCTTT’, ‘CCCGGGAAACCCGGG’, ‘CCCGGGAAAGGGAAA’,‘CCCGGGAAAGGGTTT’, ‘CCCGGGAAAGGGCCC’, ‘CCCGGGTTTAAATTT’, ‘CCCGGGTTTAAACCC’,‘CCCGGGTTTAAAGGG’, ‘CCCGGGTTTCCCAAA’, ‘CCCGGGTTTCCCTTT’, ‘CCCGGGTTTCCCGGG’,‘CCCGGGTTTGGGAAA’, ‘CCCGGGTTTGGGTTT’, ‘CCCGGGTTTGGGCCC’, ‘CCCGGGCCCAAATTT’,‘CCCGGGCCCAAACCC’, ‘CCCGGGCCCAAAGGG’, ‘CCCGGGCCCTTTAAA’, ‘CCCGGGCCCTTTCCC’,‘CCCGGGCCCTTTGGG’, ‘CCCGGGCCCGGGAAA’, ‘CCCGGGCCCGGGTTT’, ‘CCCGGGCCCGGGCCC’,‘GGGAAATTTAAATTT’, ‘GGGAAATTTAAACCC’, ‘GGGAAATTTAAAGGG’, ‘GGGAAATTTCCCAAA’,‘GGGAAATTTCCCTTT’, ‘GGGAAATTTCCCGGG’, ‘GGGAAATTTGGGAAA’, ‘GGGAAATTTGGGTTT’,‘GGGAAATTTGGGCCC’, ‘GGGAAACCCAAATTT’, ‘GGGAAACCCAAACCC’, ‘GGGAAACCCAAAGGG’,‘GGGAAACCCTTTAAA’, ‘GGGAAACCCTTTCCC’, ‘GGGAAACCCTTTGGG’, ‘GGGAAACCCGGGAAA’,‘GGGAAACCCGGGTTT’, ‘GGGAAACCCGGGCCC’, ‘GGGAAAGGGAAATTT’, ‘GGGAAAGGGAAACCC’,‘GGGAAAGGGAAAGGG’, ‘GGGAAAGGGTTTAAA’,‘GGGAAAGGGTTTCCC’, ‘GGGAAAGGGTTTGGG’,‘GGGAAAGGGCCCAAA’, ‘GGGAAAGGGCCCTTT’, ‘GGGAAAGGGCCCGGG’, ‘GGGTTTAAATTTAAA’,‘GGGTTTAAATTTCCC’, ‘GGGTTTAAATTTGGG’, ‘GGGTTTAAACCCAAA’, ‘GGGTTTAAACCCTTT’,‘GGGTTTAAACCCGGG’, ‘GGGTTTAAAGGGAAA’, ‘GGGTTTAAAGGGTTT’, ‘GGGTTTAAAGGGCCC’,‘GGGTTTCCCAAATTT’, ‘GGGTTTCCCAAACCC’, ‘GGGTTTCCCAAAGGG’, ‘GGGTTTCCCTTTAAA’,‘GGGTTTCCCTTTCCC’, ‘GGGTTTCCCTTTGGG’, ‘GGGTTTCCCGGGAAA’, ‘GGGTTTCCCGGGTTT’,‘GGGTTTCCCGGGCCC’, ‘GGGTTTGGGAAATTT’, ‘GGGTTTGGGAAACCC’, ‘GGGTTTGGGAAAGGG’,‘GGGTTTGGGTTTAAA’, ‘GGGTTTGGGTTTCCC’, ‘GGGTTTGGGTTTGGG’, ‘GGGTTTGGGCCCAAA’,‘GGGTTTGGGCCCTTT’, ‘GGGTTTGGGCCCGGG’, ‘GGGCCCAAATTTAAA’, ‘GGGCCCAAATTTCCC’,‘GGGCCCAAATTTGGG’, ‘GGGCCCAAACCCAAA’, ‘GGGCCCAAACCCTTT’, ‘GGGCCCAAACCCGGG’,‘GGGCCCAAAGGGAAA’, ‘GGGCCCAAAGGGTTT’, ‘GGGCCCAAAGGGCCC’, ‘GGGCCCTTTAAATTT’,‘GGGCCCTTTAAACCC’, ‘GGGCCCTTTAAAGGG’, ‘GGGCCCTTTCCCAAA’ ‘GGGCCCTTTCCCTTT’,‘GGGCCCTTTCCCGGG’, ‘GGGCCCTTTGGGAAA’, ‘GGGCCCTTTGGGTTT’, ‘GGGCCCTTTGGGCCC’,‘GGGCCCGGGAAATTT’, ‘GGGCCCGGGAAACCC’, ‘GGGCCCGGGAAAGGG’,‘GGGCCCGGGTTTAAA’,‘GGGCCCGGGTTTCCC’, ‘GGGCCCGGGTTTGGG’, ‘GGGCCCGGGCCCAAA’, ‘GGGCCCGGGCCCTTT’,‘GGGCCCGGGCCCGGG’。,

[0033] SEQ ID No.411-518:

[0034] 'AAATTTAAATTT', 'AAATTTAAACCC', 'AAATTTAAAGGG', 'AAATTTCCCAAA', 'AAATTTCCCTTT', 'AAATTTCCCGGG', 'AAATTTGGGAAA', 'AAATTTGGGTTT', 'AAATTTGGGCCC', 'AAACCCAAATTT', 'AAACCCAAACCC', 'AAACCCAAAGGG', 'AAACCCTTTAAA', 'AAACCCTTTCCC', 'AAACCCTTTGGG', 'AAACCCGGGAAA', 'AAACCCGGGTTT', 'AAACCCGGGCCC', 'AAAGGGAAATTT', 'AAAGGGAAACCC', 'AAAGGGAAAGGG', 'AAAGGGTTTAAA', 'AAAGGGTTTCCC', 'AAAGGGTTTGGG', 'AAAGGGCCCAAA', 'AAAGGGCCCTTT', 'AAAGGGCCCGGG', 'TTTAAATTTAAA', 'TTTAAATTTCCC', 'TTTAAATTTGGG', 'TTTAAACCCAAA', 'TTTAAACCCTTT', 'TTTAAACCCGGG', 'TTTAAAGGGAAA', 'TTTAAAGGGTTT', 'TTTAAAGGGCCC', 'TTTCCCAAATTT', 'TTTCCCAAACCC', 'TTTCCCAAAGGG', 'TTTCCCTTTAAA', 'TTTCCCTTTCCC', 'TTTCCCTTTGGG', 'TTTCCCGGGAAA', 'TTTCCCGGGTTT', 'TTTCCCGGGCCC', 'TTTGGGAAATTT', 'TTTGGGAAACCC', 'TTTGGGAAAGGG', 'TTTGGGTTTAAA', 'TTTGGGTTTCCC', 'TTTGGGTTTGGG', 'TTTGGGCCCAAA', 'TTTGGGCCCTTT', 'TTTGGGCCCGGG', 'CCCAAATTTAAA', 'CCCAAATTTCCC', 'CCCAAATTTGGG', 'CCCAAACCCAAA', 'CCCAAACCCTTT', 'CCCAAACCCGGG', 'CCCAAAGGGAAA', 'CCCAAAGGGTTT','CCCAAAGGGCCC', 'CCCTTTAAATTT', 'CCCTTAAACCC', 'CCCTTAAAGGG', 'CCCTTTCCCAAA', 'CCCTTTCCCTTT', 'CCCTTTCCCGGG', 'CCCTTTGGGAAA', 'CCCTTTGGGTTT', 'CCCTTTGGGCCC', 'CCCGGGAAATTT', 'CCCGGGAAACCC', 'CCCGGGAAAGGG', 'CCCGGGTTTAAA', 'CCCGGGTTTCCC', 'CCCGGGTTGGG', 'CCCGGGCCCAAA', 'CCCGGGCCCTTT', 'CCCGGGCCCGGG', 'GGGAAATTTAAA', 'GGGAAATTTCCC', 'GGGAAATTTGGG', 'GGGAAACCCAAA', 'GGGAAACCCTTT', 'GGGAACCCGGG', 'GGGAAAGGGAAA', 'GGGAAAGGGTTT', 'GGGAAAGGGCCC', 'GGGTTTAAATTT', 'GGGTTTAAACCC', 'GGGTTTAAAGGG', 'GGGTTTCCCAAA', 'GGGTTTCCCTTT', 'GGGTTTCCCGGG', 'GGGTTTGGGGAAA', 'GGGTTTGGGTTT', 'GGGTTTGGGGCCC', 'GGGCCCAAATTT', 'GGGCCCAAACCC', 'GGGCCCAAAGGG', 'GGGCCCTTTTAAA', 'GGGCCCTTTCCC', 'GGGCCCTTTGGG', 'GGGCCCGGGAAA', 'GGGCCCGGGTTT', 'GGGCCCGGGCCC'. ,

[0035] In some implementations, the reaction conditions for step (4) of denaturation-annealing-extension include: denaturation: 85-95℃ for 5-15s, annealing: 30-50℃ for 5-15s, extension: 50-65℃ for 5-15s, 3-8 cycles.

[0036] In some implementations, in step (5), the cleaning solution required for the cleaning contains 0.5-2 M sodium carbonate.

[0037] In some implementations, in step (7), the synthesized product may be stripped from the solid support using, but is not limited to, 6-8 M urea or endonuclease V, and the stripping method may be adjusted according to the immobilization method.

[0038] In some embodiments, the synthesis method of the present invention as described above can be used, but is not limited to, the synthesis of nucleic acid molecular variants.

[0039] This invention also provides the application of a nucleic acid molecular variant as described above in data storage (including but not limited to data storage). The optimal nucleic acid molecular variant for data storage consists of homotrimers.

[0040] Compared with the prior art, the beneficial effects of the present invention are:

[0041] 1. This invention proposes using homopolymers of nucleotides instead of mononucleotides as the basic unit for information encoding. Because this can improve the redundancy and robustness of information encoding at the molecular structure level, it can significantly improve the accuracy of nucleic acid molecules for information storage. Its structure, different from that of natural molecules, also makes it easier to identify.

[0042] 2. This invention provides multiple polymerase variants and develops nucleic acid molecule synthesis methods based on them. Template-dependent polymerases are used to replace TdT enzymes, which greatly improves accuracy. Secondary structure problems are solved through high-temperature reactions to achieve the synthesis of long-fragment nucleic acid molecules.

[0043] 3. All embodiments of the present invention do not require expensive modified nucleotide substrates, nor do they require the reversible terminator necessary in methods such as TdT enzymes as substrates. Instead, they utilize the precise control provided by the base complementarity between primers and templates and the fidelity of polymerase variants to achieve the incorporation of single-step nucleotide homopolymers, thereby achieving controlled synthesis of ordered nucleic acid molecules.

[0044] 4. This invention significantly improves the efficiency of nucleic acid molecule synthesis, reaching or even exceeding the efficiency of classical chemical synthesis methods (≥99.50%). When used for data storage, its accuracy far surpasses that of conventional nucleic acid molecules that rely on single nucleotide encoding.

[0045] 5. This invention significantly reduces the cost of nucleic acid molecule synthesis and its use as a storage medium, and has the potential to synthesize long-fragment nucleic acid molecules, opening up new ideas and methods for the field of nucleic acid molecule biosynthesis technology. Attached Figure Description

[0046] Figure 1The diagram shows the structure and synthesis process of nucleic acid molecule variants. In this diagram, A is a comparison of the sequence structure of nucleic acid molecule variants and conventional nucleic acid molecules; B is a diagram showing the specific composition of the nucleotide homodimers, homotrimers, and homotetramers of the nucleic acid molecule variants; C is a flowchart of the synthesis method of nucleic acid molecule variants based on a series of templates; and D is a structural diagram of the template.

[0047] Figure 2 A comparison of high-throughput sequencing results and corrected results for a conventional nucleic acid molecule composed of single nucleotides and a nucleic acid molecule variant composed of homotrimers encoding "Information 1";

[0048] Figure 3 A comparison of high-throughput sequencing results and corrected results for a conventional nucleic acid molecule composed of single nucleotides and a nucleic acid molecule variant composed of homotrimers encoding "Information 2";

[0049] Figure 4 A comparison of high-throughput sequencing results and corrected results for a conventional nucleic acid molecule composed of single nucleotides and a nucleic acid molecule variant composed of homotrimers encoding "Information 3";

[0050] Figure 5 A statistical chart comparing the accuracy of synthesis and data storage for conventional nucleic acid molecules composed of single nucleotides and nucleic acid molecule variants composed of homotrimers.

[0051] Figure 6 This is a schematic diagram of the polymerase variant modification used in this invention;

[0052] Figure 7 The image shows the detection results of the enzymatically synthesized product in Example 2. In the image, A is a 15% urea-polyacrylamide gel electrophoresis image, and B is the result obtained from the analysis and statistics based on the gel electrophoresis image.

[0053] Figure 8 This is a 15% urea-polyacrylamide gel electrophoresis image of the enzymatically synthesized product of Example 3;

[0054] Figure 9 This is a 15% urea-polyacrylamide gel electrophoresis image of the enzymatically synthesized product of Example 4.

[0055] Figure 10 The images show the detection results of the nucleic acid molecular variant synthesized in Example 4, where A is the Sanger sequencing result and B is the high-throughput sequencing result.

[0056] Figure 11 This is a 15% urea-polyacrylamide gel electrophoresis image of the enzymatically synthesized product of Example 5;

[0057] Figure 12This is a statistical graph showing the accuracy of the nucleic acid molecular variants synthesized in Example 5 during synthesis and encoding.

[0058] Figure 13 The high-throughput sequencing results for the nucleic acid molecular variants synthesized in Example 5 are shown below. In this diagram, A represents the high-throughput sequencing results for the nucleic acid molecular variant encoding 1T, B represents the high-throughput sequencing results for the nucleic acid molecular variant encoding 2D, C represents the high-throughput sequencing results for the nucleic acid molecular variant encoding 3E, D represents the high-throughput sequencing results for the nucleic acid molecular variant encoding 4D, and E represents the high-throughput sequencing results for the nucleic acid molecular variant encoding 5S. Detailed Implementation

[0059] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0060] 1. This invention utilizes homopolymers of nucleotides instead of individual nucleotides as the basic unit of information encoding for DNA data storage. It provides a nucleic acid molecule variant composed of homopolymers of nucleotides, with the sequence structure as follows: Figure 1 As shown in A in the diagram.

[0061] When DNA is used as a medium for information storage, information is typically represented by a single nucleotide or a transformation of nucleotides. However, errors can occur during synthesis and reading; the deletion, insertion, or mutation of a single nucleotide can lead to deviations in the encoded information, requiring complex error correction procedures. This invention uses homopolymers of nucleotides, such as homodimers, homotrimers, homotetramers, homopentamers, homohexamers, homoheptamers, and homooctamers, instead of single nucleotides as the basic unit of information encoding. The sequence structure of the nucleic acid molecule variants composed of these homopolymers is shown below. Figure 1 As shown in B in the diagram. Even if a nucleotide is mutated, the correct nucleotide can be deduced based on the homopolymer coding principle and neighboring nucleotides. This structure does not exist in nature and can effectively distinguish between artificially synthesized and naturally occurring nucleic acid molecules, while also avoiding contamination and confusion.

[0062] 2. A series of polymerase variants were obtained, capable of efficiently and precisely incorporating homopolymers of nucleotides under template guidance. The accurate synthesis of nucleic acid molecules in nature relies on template-guided polymerase replication or transcription. It is estimated that even without proofreading activity, the error rate of polymerase synthesis is only about one in ten thousand, far exceeding the 99% of chemical synthesis methods. Adding only one target nucleotide per step during synthesis, instead of a mixture of four nucleotides, further improves the accuracy of synthesis. Typically, polymerases require primers and templates to be at least 18 bases complementary, meaning the template must contain at least 18 nucleotides. If template-dependent addition of a single nucleotide to the primer is required, a template containing at least 19 nucleotides is needed. To synthesize any nucleic acid molecule, four nucleotides are required. 19 There are several templates required. However, if a nucleic acid molecule variant composed of homopolymers is synthesized, the number of templates required is significantly reduced. For example, to synthesize a homotrimeric nucleic acid molecule variant, if the primer and template have 18 complementary bases, and one homotrimer is added, meaning the core region of the template has 21 nucleotides, the required number of templates is 4 × 3. 21 / 3-1 =2916 kinds, far less than 4 19 This invention develops polymerase variants, including TaqΔN-TM, Dpo4ΔC, and Dpo4ΔC-CL7, which enable accurate synthesis of nucleic acid molecules even with short complementary regions. Even when the primer and template have only 9 complementary bases (i.e., only 12 nucleotides in the template core region), three nucleotides can be accurately added to the 3' end of the primer. This allows for the precise addition of three nucleotides to the primer using only 4 × 3... 12 / 3-1 With 108 templates, any homotrimeric nucleic acid molecule variant can be synthesized.

[0063] 3. Template-dependent nucleic acid molecule synthesis methods: skip synthesis. A schematic diagram of the synthesis method is shown below. Figure 1 As shown in C.

[0064] Design a template library where each template core region contains no more than 21 nucleotides, and the library covers all combinations of homopolymers of nucleotides with a maximum number of 21 nucleotides. A schematic diagram of the template structure is shown below. Figure 1 As shown in D in the diagram.

[0065] The skipping method utilizes polymerase variants to synthesize ordered nucleic acid molecules through a cyclic reaction of high-temperature denaturation, low-temperature annealing, and optimal-temperature extension, as well as base complementarity between primers and templates. High-temperature denaturation unlocks secondary structures. Each synthesis step uses a different template, essentially meaning the 3' end of the primer skips between different templates, hence the name "skipping method."

[0066] 3.1 Design a starting primer (N) with a length of 22–30 nucleotides;

[0067] 3.2 Immobilize the starting primers from 3.1 onto a solid support;

[0068] 3.3 Add reaction buffer, target nucleotide molecule, template, and any one of the polymerase variants mentioned above to the product obtained in 3.2;

[0069] 3.4 A given number of target nucleotide molecules are added to the 3' end of the primer through denaturation, annealing, and extension. Utilizing base complementarity, the fidelity of polymerase variants, and high-purity nucleotide substrates, the correct number of nucleotide molecules can be introduced to the 3' end of the primer. To improve efficiency, the denaturation-annealing-extension process in the single-step synthesis reaction can be repeated in multiple cycles.

[0070] 3.5 Wash away the template, polymerase variant, and unreacted target nucleotide molecules to obtain a primer with one homopolymer added (N+1).

[0071] 3.6 Repeat steps 3.3-3.5 until synthesis is complete;

[0072] 3.7 Remove the synthesized primers from the solid support.

[0073] In one specific embodiment of the present invention

[0074] (1) The template described in 3.1 is a series of single-stranded oligonucleotides that are complementary to the primers by 16, 15, 12, 9 or 6 bases, which can guide the synthesis of any nucleic acid variant molecule. In this embodiment, only the template required for this embodiment is listed. Other templates are not listed but are included in the template library.

[0075] (2) 3.2 The solid-phase support is streptavidin magnetic beads, and the starting primer is biotin and FAM modified.

[0076] (3) The reaction system described in step 3.3 consists of 40 pmol primers, 20-80 pmol template, 400 μM dATP / dTTP / dCTP / dGTP, 10-40 pmol polymerase variant, and 1× reaction buffer (10-40 mM Tris (pH 7.5-8.0), 40-60 mM KCl, 1-10 mM MgCl2, 1-10 mM DTT), with a total volume of 25 μL.

[0077] (4) The polymerase variant described in step 3.3 is TaqΔN-TM, Dpo4ΔC or Dpo4ΔC-CL7.

[0078] (5) The reaction conditions described in step 3.4 are 85-95℃ for 5-15s, 30-50℃ for 5-15s, 50-65℃ for 5-15s, for 5 cycles.

[0079] (6) The cleaning solution described in step 3.5 is a Tris buffer solution containing 0.5-2 M sodium carbonate.

[0080] (7) In step 3.7, 8 M urea is used to peel the synthesized product off the solid support.

[0081] In another specific embodiment of the present invention

[0082] (1) The template described in 3.1 is a series of single-stranded oligonucleotides that are 12 or 9 bases complementary to the primers and can guide the synthesis of any nucleic acid variant molecule. In this embodiment, only the templates required for this embodiment are listed; other templates are not listed but are included in the template library.

[0083] (2) 3.2 The solid-phase support is streptavidin magnetic beads, and the starting primer is biotin and FAM modified.

[0084] (3) The reaction system described in step 3.3 consists of 40 pmol primers, 20-80 pmol template, 400 μM dATP / dTTP / dCTP / dGTP, 25-30% glycerol, 10-40 pmol polymerase variant, and 1× reaction buffer (10-40 mM Tris (pH 7.5-8.0), 40-60 mM KCl, 1-10 mM MgCl2, 1-10 mM DTT), with a total volume of 25 μL.

[0085] (4) The polymerase variant described in step 3.3 is Dpo4ΔC-CL7.

[0086] (5) The reaction conditions described in step 3.4 are 85-95℃ for 5-15s, 30-50℃ for 5-15s, 50-65℃ for 5-15s, for 5 cycles.

[0087] (6) The cleaning solution described in step 3.5 is a Tris buffer solution containing 0.5-2 M sodium carbonate.

[0088] (7) In step 3.7, 8 M urea is used to peel the synthesized product off the solid support.

[0089] Example 1

[0090] Accuracy comparison of nucleic acid molecule variants and conventional nucleic acid molecules in data storage

[0091] When used as an information storage medium, nucleic acid molecules composed of homopolymers of nucleotides have higher information fidelity than conventional nucleic acid molecules composed of single nucleotides.

[0092] 1. The accuracy of information extraction was compared by encoding the same information using both ordinary mononucleotides and homotrimers. Table 1 shows three pairs of sequences encoding information 1, information 2, and information 3, respectively.

[0093] Table 1. Comparison of sequences encoding the same information in mononucleotides and homopolymers.

[0094]

[0095] 2. Synthesize three pairs of nucleic acid molecules composed of mononucleotides or homotrimers of nucleotides.

[0096] Nucleic acid molecules composed of single nucleotides:

[0097] SEQ ID No.4:

[0098] 5'-GAGAGCAGCTATGCAGCTT AGTGCAGTAC TCGAGCACCACCACCACCACC-3';

[0099] SEQ ID No. 5:

[0100] 5'-GAGAGCAGCTATGCAGCTT CATGCTACGA TCGAGCACCACCACCACCACC-3';

[0101] SEQ ID No. 6:

[0102] 5'-GAGAGCAGCTATGCAGCTT GCTCTGCGCA TCGAGCACCACCACCACCACC-3'.

[0103] Nucleic acid molecules composed of homotrimers of nucleotides:

[0104] SEQ ID No. 7:

[0105] 5'-GAGAGCAGCTATGCAGCTT AAAGGTTTGGGCCCAAAGGGTTTAAACCC TCGAGCACCACCACCACCACC-3';

[0106] SEQ ID No. 8:

[0107] 5'-GAGAGCAGCTATGCAGCTT CCCAAATTTGGGCCCTTTAAACCCGGGAAA TCGAGCACCACCACCACCACC-3';

[0108] SEQ ID No. 9:

[0109] 5'-GAGAGCAGCTATGCAGCTT GGGCCCTTTCCCTTTGGGCCCGGGCCCAAATCGAGCACCACCACCACCACC-3'.

[0110] 3. Sample preparation and high-throughput sequencing, the specific steps are as follows:

[0111] a. Prepare the reaction systems according to Tables 2 and 3 respectively;

[0112] Table 2. Fragment I PCR System

[0113]

[0114] Note: Universal primer T7Terminator: 5'-GCTAGTTATTGCTCAGCGG-3'; SEQ ID No. 84.

[0115] Table 3. Fragment II PCR System

[0116]

[0117] Note: T7 promoter: 5'-TAATACGACTCACTATAGGG-3'; SEQ ID No. 85.

[0118] Downstream primer: 5'-AAGCTGCATAGCTGCTCTCCTTCTTAAAG-3'; SEQ ID No. 86.

[0119] b. Following the reaction procedure shown in Table 4, PCR products of fragment I and fragment II were obtained respectively;

[0120] Table 4 PCR reaction procedure

[0121]

[0122] c. Perform a splicing reaction on fragment I and fragment II, prepare the reaction system as shown in Table 5, and carry out the reaction according to the procedure in Table 4 to obtain the spliced ​​product.

[0123] Table 5. Composite Reaction System

[0124]

[0125] 4. Product recovery and testing, the specific procedures are as follows:

[0126] The product obtained in step 3c was recovered by gel extraction, and the recovered product was subjected to high-throughput sequencing.

[0127] 5. Data Analysis

[0128] To address the vulnerability of single-nucleotide-based data encoding to errors introduced during synthesis, storage, and sequencing, this invention proposes replacing single nucleotides with homopolymers, such as homotrimers (e.g., AAA) and homotetramers (e.g., AAAA), as the basic coding unit to achieve high error tolerance and robust data recovery. Even with synthetic errors, the correct nucleotide can be derived from its homopolymer structure, enabling information correction. High-throughput sequencing results and corrections are shown below. Figures 2-4 As shown. Figure 2 The images, from bottom to top, show the sequencing results of a synthesized nucleic acid molecule variant composed of homotrimers, the corrected results, and the sequencing results of a synthesized nucleic acid molecule composed of single nucleotides; the percentages of correct sequences are 97.71%, 100.00%, and 99.59%, respectively. Figure 3 The images, from bottom to top, show the sequencing results of a synthesized nucleic acid molecule variant composed of homotrimers, the corrected results, and the sequencing results of a synthesized nucleic acid molecule composed of single nucleotides; the percentages of correct sequences are 96.17%, 100.00%, and 99.87%, respectively. Figure 4 The images, from bottom to top, show the sequencing results of a synthesized nucleic acid molecule variant composed of homotrimers, the corrected results, and the sequencing results of a synthesized nucleic acid molecule composed of single nucleotides; the percentages of correct sequences are 97.76%, 100.00%, and 99.39%, respectively.

[0129] In one typical experiment, the average stepwise accuracy for single nucleotides was 99.96%, while the average stepwise accuracy for homotrimers was 99.77%. However, by analyzing specific sequences, it was found that each erroneous homotrimer contained only one nucleotide deletion or substitution. Based on the structure of the homotrimer, the correct homotrimer sequence could be deduced from the other two nucleotides, thus correcting the sequencing results. After correction, the coding accuracy of nucleic acid molecule variants composed of homotrimers could reach 100.00%. However, nucleic acid molecules composed of single nucleotides cannot be corrected in this way. Figure 5 As shown, although the synthesis accuracy of homotrimers is lower than that of single nucleotides, the coding accuracy is higher.

[0130] Example 2

[0131] Screening of polymerase variants

[0132] 1. Construct and express wild-type and variant polymerases

[0133] a. Construct recombinant plasmids and clone the genes encoding TaqΔN-TM, Dpo4ΔC, and Dpo4ΔC-CL7 into the pET28a vector. The amino acid sequences are shown in SEQ ID No. 1 (TaqΔN-TM), SEQ ID No. 2 (Dpo4ΔC), and SEQ ID No. 3 (Dpo4ΔC-CL7), respectively. A schematic diagram of the modification is shown below. Figure 6 As shown.

[0134] Recombinant plasmids were constructed, and the genes encoding TaqΔN, Dpo4, pfu-AG, TbrΔN, and TbrΔN-TM were cloned into the pET28a vector. The amino acid sequences are shown in SEQ ID No. 10 (TaqΔN), SEQ ID No. 11 (Dpo4), SEQ ID No. 12 (pfu-AG), SEQ ID No. 13 (TbrΔN), and SEQ ID No. 14 (TbrΔN-TM), respectively.

[0135] SEQ ID No. 1:

[0136] .

[0137] SEQ ID No.2:

[0138] MIVLFVDFDYFYAQVEEVLNPSLKGKPVVVCVFSGRFEDSGAVATANYEARKFGVKAGIPIVEAKKILPNAVYLPMRKEVYQQVSSRIMNLLREYSEKIEIASIDEAYLDISDKVRDYREAYNLGLEIKNKILEKEKITVTVGISKNKVFAKIAADMAKPNGIKVIDDEEVKRLIRELDIADVPGIGNITAEKLKKLGINKLVDTLSIEFDKLKGMIGEAKAKYLISLARDEYNEPIRTRLEHHHHHH.

[0139] SEQ ID No.3:

[0140] MIVLFVDFDYFYAQVEEVLNPSLKGKPVVVCVFSGRFEDSGAVATANYEARKFGVKAGIPIVEAKKILPNAVYLPMRKEVYQQVSSRIMNLLREYSEKIEIASIDEAYLDISDKVRDYREAYNLGLEIKNKILEKEKITVTVGISKNKVFAKIAADMAKPNGIKVIDDEEVKRLIRELDIADVPGIGNITAEKLKKLGINKLVDTLSIEFDKLKGMIGEAKAKYLISLARSNSLEVLFQGPTAAAASKSNEPGKATGEGKPVNNKWLNNAGKDLGSPVPDRIANKLRDKEFESFDDFRETFWEEVSKDPELSKQFSRNNNDRMKVGKAPKTRTQDVSGKRTSFELNHQKPIEQNGGVYDMDNISVVTPKRNIDIELEHHHHHHHH.

[0141] SEQ ID No.10:

[0142] 。

[0143] SEQ ID No.11:

[0144] MIVLFVDFDYFYAQVEEVLNPSLKGKPVVVCVFSGRFEDSGAVATANYEARKFGVKAGIPIVEAKKILPNAVYLPMRKEVYQQVSSRIMNLLREYSEKIEIASIDEAYLDISDKVRDYREAYNLGLEIKNKILEKITVVGISNKVFAKIAADMAKPNGIKIDDEEVKRLIRELDI ADVPGIGNITAEKLKKLGINLVDTLSIEFDKLKGMIGEAKAKYLISLARDEYNEPIRTRVRSIGRIVTMKRNSNLEEIKPYLFRAIEESYYKLDKRIPKAIHVVAVTEDLDIVSRGRTFPHGISKETAYSESVKLLQKILEEDERKIRRIGVRFSKFIEAIGLDKFFDTLEHHHHHH。

[0145] SEQ ID No.12:

[0146] 。

[0147] SEQ ID No.13:

[0148] MHHHHHHGSAAEEAPWPPPEGAFLGFRLSRPEPMWAELLSLAASAKGRVYRAEAPHKALSDLKEIRGLLAKDLAVLALREGLGLPPTDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERALLAERLYENLLSRLKGEEKLLWLYEEVEKPLSRVLAHMEATGVRLDVPYLRALSLEVAAEMGRLEEEVFRLAGHPFNLNSRDQLERVLFDELGLPPIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELAKLKGTYIDPLPALVHPRTGRLHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFVAEEGYLLVALDYSQIELRVLAHLSGDENLIRVFQEGRDIHTQTASWMFGLPAEAIDPLRRRAAKTINFGVLYGMSAHRLSQELGIPYEEAVAFIDRYFQSYPKVKAWIERTLEEGRQRGYVETLFGRRRYVPDLNARVKSVREAAERMAFNMPVQGTAADLMKLAMVRLFPRLPEVGARMLLQVHDELLLEAPKERAEEAAALAKEVMEGVWPLAVPLEVEVGIGEDWLSAKG。

[0149] SEQ ID No.14:

[0150] .

[0151] b. Transform the plasmid into E. coli BL21(DE3). Inoculate a single clone into LB medium and culture until the OD600 reaches 0.6. Then add IPTG to a final concentration of 0.4 mM and induce overnight at 25°C.

[0152] c. Purification and preparation of wild-type and variant polymerase: Cells were collected and sonicated, then heated in a 70°C water bath for 15 min. After centrifugation, the supernatant was collected and purified using immobilized metal affinity chromatography (Ni-NTA resin), followed by further purification using Superdex 200 Increase 10 / 300 GL. The wild-type and variant polymerase were desalted, concentrated by ultrafiltration, and the buffer was replaced with 1× enzyme stock solution (30 mM Tris-HCl pH 8.0, 1 mM DTT, 0.1 mM EDTA, 0.1% Tween-20, 50% glycerol). The solution was stored at -20°C for later use.

[0153] 2. Design and synthesize starting primers and templates.

[0154] The starting primer sequence is:

[0155] SEQ ID No. 15: 5'Biotin-GGAGAGCAGCT(6-FAM)ATGCAGCTTAGCAAGGGC-3'.

[0156] The template sequence is:

[0157] SEQ ID No.16: 5'-ctcgagTTTGCCCTTGCTAAGCTGCATAGCcatatg-3';

[0158] SEQ ID No.17: 5'-ctcgagAAAGCCCTTGCTAAGCTGCATAGCcatatg-3';

[0159] SEQ ID No.18: 5'-ctcgagCCCGCCCTTGCTAAGCTGCATAGCcatatg-3';

[0160] SEQ ID No. 19: 5'-ctcgacGGGGCCCTTGCTAAGCTGCATAGCcatatg-3'.

[0161] They all have 21 base complements to the primers, and can guide the addition of homotrimers AAA, TTT, GGG, or CCC to the 3' end of the primers, respectively.

[0162] 3. The biotin-modified starter primer (SEQ ID No. 15) was bound to streptavidin magnetic beads. The specific steps are as follows:

[0163] a. For each reaction, take 25 μL of streptavidin magnetic beads and place them in a 1.5 mL centrifuge tube for later use; before use, place the tube on a magnetic rack for 1 min to separate and remove the supernatant;

[0164] b. Add 500 μL 1×TBS and gently resuspend the streptavidin magnetic beads by pipetting; place on a magnetic rack for separation for 30 s, remove the supernatant, and complete one washing step; then wash twice more according to the aforementioned washing steps;

[0165] c. Resuspend the magnetic beads in 50 μL of 2× binding buffer;

[0166] 2× binding buffer: 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 2 M NaCl, 0.01%-0.1% Tween-20;

[0167] d. Add 4 μL of biotin-labeled primer (SEQ ID No. 15) (10 μM), then add 46 μL of ultrapure water, thoroughly pipette to suspend, place on a rotary mixer, and incubate at room temperature for 30 min;

[0168] e. Place the centrifuge tubes on a magnetic rack and centrifuge for 1 minute, then remove the supernatant;

[0169] d. Add 1 ml of 1× TBS to the separated magnetic beads and thoroughly resuspend them by blowing. Place the centrifuge tube on a magnetic rack and separate for 1 min. Remove the supernatant. Repeat the washing process twice more and set aside for use.

[0170] 4. Enzyme-catalyzed synthesis reaction, the specific steps are as follows:

[0171] a. Transfer the primers that have been bound to the magnetic beads into PCR tubes and add the reaction reagents. Each reaction is prepared according to the system shown in Table 6 below.

[0172] Table 6 Enzyme-catalyzed reaction system

[0173]

[0174] 10×Reaction Buffer: 400 mM Tris (pH 8.0), 60 mM KCL, 1 mM MgCL2, 10 mMDTT, 2.5% glycerol.

[0175] b. Place the PCR tube in the PCR instrument and react according to the procedure shown in Table 7;

[0176] Table 7. Procedures for Enzyme-Catalyzed Synthesis Reactions

[0177]

[0178] c. After the reaction is complete, remove the PCR tube, place it on a magnetic rack for 1 minute to separate, and remove the supernatant;

[0179] d. Add 200 μL of washing buffer, thoroughly pipette to resuspend the magnetic beads, and let stand for 2 min. Place the PCR tube on a magnetic rack for 1 min to separate, and remove the supernatant. Wash three more times with 1× reaction buffer before proceeding to the next step.

[0180] Washing buffer: 20 mM Tris (pH 8.0), 150 mM NaCl, 1 M sodium carbonate, 10% glycerol.

[0181] e. Add 5 μL of 8 M urea, heat at 95°C for 10 min, and peel the synthesized product off the magnetic beads.

[0182] 5. Detection of enzyme-catalyzed synthesis products, the specific procedures are as follows:

[0183] The enzymatically synthesized product was detected by 15% urea-polyacrylamide gel electrophoresis (voltage: 220 V, 1 h).

[0184] 6. Results and Analysis

[0185] The 15% urea-polyacrylamide gel electrophoresis results of the enzymatically synthesized products of this invention are as follows: Figure 7 As shown in A, Figure 7 B in the text is based on Figure 7 The statistical table is obtained by performing statistics on A in the table. Figure 7 It is evident that only TaqΔN-TM, Dpo4ΔC, and Dpo4ΔC-CL7 can perform rigorous template-dependent synthesis of nucleotide homopolymers, with no template-independent synthesis. In contrast, other polymerase wild-types or variants can perform template-independent synthesis. Subsequent experiments will preferentially select the three polymerase variants, TaqΔN-TM, Dpo4ΔC, and Dpo4ΔC-CL7.

[0186] Example 3

[0187] Determination of the optimal template for enzymatic synthesis

[0188] 1. Construct and express polymerase variants. The specific steps are the same as step 1 in Example 2. The difference is that this example only constructs and expresses TaqΔN-TM, Dpo4ΔC, and Dpo4ΔC-CL7.

[0189] 2. Design and synthesize starting primers and templates.

[0190] The starting primer sequence is:

[0191] SEQ ID No. 15: 5'Biotin-GGAGAGCAGCT(6-FAM)ATGCAGCTTAGCAAGGGC-3'.

[0192] The template sequence is:

[0193] 16 complementary bases: (used to synthesize nucleic acid molecule variants composed of homotetramers of nucleotides)

[0194] First step reaction template: SEQ ID No. 20: 5'-ctcgagTTTTGCCCTTGCTAAGCTGCcatatg-3';

[0195] Second step reaction template: SEQ ID No. 21: 5'-ctcgagCCCCTTTTGCCCTTGCTAAGcatatg-3';

[0196] Third step reaction template: SEQ ID No. 22: 5'-ctcgagAAAACCCCTTTTGCCCTTGCcatatg-3';

[0197] Fourth step reaction template: SEQ ID No. 23: 5'-ctcgagCCCCAAAACCCCTTTTGCCCcatatg-3';

[0198] Fifth step reaction template: SEQ ID No. 24: 5'-ctcgacGGGGCCCCAAAACCCCTTTTcatatg-3'.

[0199] 15 complementary bases: (used to synthesize nucleic acid molecule variants composed of homotrimers of nucleotides)

[0200] First step reaction template: SEQ ID No. 25: 5'-gacctcgagTTTGCCCTTGCTAAGCTGcatatg-3';

[0201] Second step reaction template: SEQ ID No. 26: 5'-gacctcgagCCCTTTGCCCTTGCTAAGcatatg-3';

[0202] Third step reaction template: SEQ ID No. 27: 5'-gacctcgagAAACCCTTTGCCCTTGCTcatatg-3';

[0203] Fourth step reaction template: SEQ ID No. 28: 5'-gacctcgagCCCAAACCCTTTGCCCTTcatatg-3';

[0204] Fifth step reaction template: SEQ ID No. 29: 5'-gacctcgacGGGCCCAAACCCTTTGCCgatatg-3'.

[0205] 12 complementary bases: (used to synthesize nucleic acid molecule variants composed of homotrimers of nucleotides)

[0206] First step reaction template: SEQ ID No. 30: 5'-gacctcgagTTTGCCCTTGCTAAGcatatg-3';

[0207] Second step reaction template: SEQ ID No. 31: 5'-gacctcgagCCCTTTGCCCTTGCTcatatg-3';

[0208] Third step reaction template: SEQ ID No. 32: 5'-gacctcgagAAACCCTTTGCCCTTcatatg-3';

[0209] Fourth step reaction template: SEQ ID No. 33: 5'-gacctcgagCCCAAACCCTTTGCCcatatg-3';

[0210] Fifth step reaction template: SEQ ID No. 34: 5'-gacctcgacGGGCCCAAACCCTTTgatatg-3'.

[0211] Nine complementary bases: (used to synthesize nucleic acid molecule variants composed of homotrimers of nucleotides)

[0212] First step reaction template: SEQ ID No. 35: 5'-ctcgagTTTGCCCTTGCTcatatg-3';

[0213] Second step reaction template: SEQ ID No. 36: 5'-ctcgagCCCTTTGCCCTTcatatg -3';

[0214] Third step reaction template: SEQ ID No. 37: 5'-ctcgagAAACCCTTTGCCcatatg -3';

[0215] Fourth step reaction template: SEQ ID No. 38: 5'-ctcgagCCCAAACCCTTTcatatg-3';

[0216] Fifth step reaction template: SEQ ID No.39: 5'-ctcgacGGGCCCAAACCCgatatg -3'.

[0217] Six complementary bases: (used to synthesize nucleic acid molecule variants composed of homotrimers of nucleotides)

[0218] First step reaction template: SEQ ID No. 40: 5'-ctcgagTTTGCCCTTcatatg-3';

[0219] Second step reaction template: SEQ ID No. 41: 5'-ctcgagCCCTTTGCCcatatg -3';

[0220] Third step reaction template: SEQ ID No. 42: 5'-ctcgagAAACCCTTTcatatg-3';

[0221] Fourth step reaction template: SEQ ID No. 43: 5'-ctcgagCCCAAACCCcatatg-3';

[0222] Fifth step reaction template: SEQ ID No. 44: 5'-ctcgacGGGCCCAAAgatatg -3'.

[0223] The protective bases in the template sequences listed herein and below can also be replaced by nucleotide homopolymers, allowing the templates to be synthesized using the methods described in this invention. Furthermore, some core regions in the templates listed herein and below are not entirely composed of nucleotide homopolymers (e.g., SEQ ID No. 35, SEQ ID No. 36, SEQ ID No. 37). This is necessary for complementarity with the initial primers to verify that the invention can precisely add nucleotide homopolymers. This situation can be avoided by designing the initial primers with a 3' end composed of nucleotide homopolymers.

[0224] 3. The biotin-modified starter primer (SEQ ID No. 15) is bound to streptavidin magnetic beads, and the specific steps are the same as step 3 in Example 2.

[0225] 4. The first step, the enzymatic synthesis reaction, has the following specific steps:

[0226] a. Transfer the primers that have been bound to the magnetic beads into PCR tubes and add the reaction reagents. Each reaction is prepared according to the system shown in Table 8 below.

[0227] Table 8. First-step enzyme-catalyzed reaction system

[0228]

[0229] 10×Reaction Buffer: 400 mM Tris (pH 8.0), 60 mM KCL, 1 mM MgCL2, 10 mMDTT, 2.5% glycerol.

[0230] b. Place the PCR tube in the PCR instrument and react according to the procedure shown in Table 7;

[0231] c. After the reaction is complete, remove the PCR tube, place it on a magnetic rack for 1 minute to separate, and remove the supernatant;

[0232] d. Add 200 μL of washing buffer, thoroughly resuspend the magnetic beads by pipetting, and let stand for 2 min. Place the PCR tube on a magnetic rack for 1 min to separate, and remove the supernatant. Wash three more times with 1× reaction buffer before proceeding to the next step.

[0233] Washing buffer: 20 mM Tris (pH 8.0), 150 mM NaCl, 1 M sodium carbonate, 10% glycerol.

[0234] 5. Proceed to the second enzymatic synthesis reaction. The specific steps are the same as the first enzymatic synthesis reaction, except that the magnetic beads-starting primers in Table 8 are replaced with the products of the first step reaction, the template SEQ ID No. 20 / 25 / 30 / 35 / 40 of the first step reaction is replaced with the template SEQ ID No. 21 / 26 / 31 / 36 / 41 of the second step reaction, and dATP is replaced with dGTP. Continue in this manner until all five steps of the reaction are completed.

[0235] 6. Detection of enzyme-catalyzed synthesis products, the specific procedures are as follows:

[0236] The products of each enzymatic synthesis step were detected by 15% urea-polyacrylamide gel electrophoresis (voltage: 220 V, 1 h).

[0237] 7. Results and Analysis

[0238] The 15% urea-polyacrylamide gel electrophoresis results of the enzymatically synthesized product in this embodiment are as follows: Figure 8 As shown. The efficiency of three DNA polymerase variants, TaqΔN-TM, Dpo4ΔC, and Dpo4ΔC-CL7, in accurately synthesizing homotrimeric or homotetrameric nucleic acid variants was compared, as well as the effect of primer-template complementary region length on the accurate synthesis of homotrimeric nucleic acid variants by TaqΔN-TM and Dpo4ΔC-CL7. Figure 8 As can be seen, Dpo4ΔC-CL7, TaqΔN-TM, and Dpo4ΔC can all synthesize homotrimeric or homotetrameric nucleic acid variants, but the polymerase variant Dpo4ΔC-CL7 exhibits higher synthesis efficiency and a cleaner background. Compared to homotetrameric nucleic acid variants, homotrimeric nucleic acid variants are synthesized more efficiently in consecutive multi-step synthesis. The complementary region length between the primer and template has a significant impact on synthesis efficiency. Besides templates with a complementary region length of 6 nucleotides, templates with lengths of 15, 12, and 9 nucleotides can effectively guide the synthesis of homotrimeric nucleic acid variants. Therefore, in subsequent examples, templates with a complementary region length of 12 or 9 nucleotides, i.e., a core region of 5 or 4 homotrimeric units, are preferred for synthesizing homotrimeric nucleic acid variants.

[0239] Example 4

[0240] A nucleic acid molecular variant consisting of 10 homotrimers (30 nucleotides) was synthesized stepwise through a ten-step cycle.

[0241] 1. Construct and express the Dpo4 polymerase variant. The specific steps are the same as step 1 in Example 2. The difference is that this example only constructs and expresses Dpo4ΔC-CL7.

[0242] 2. Design and synthesize starting primers and templates.

[0243] The starting primer sequence is:

[0244] SEQ ID No. 15: 5'Biotin-GGAGAGCAGCT(6-FAM)ATGCAGCTTAGCAAGGGC-3'.

[0245] The template sequence is:

[0246] First step reaction template: SEQ ID No. 35: 5'-ctcgagTTTGCCCTTGCTcatatg-3';

[0247] Second step reaction template: SEQ ID No. 31: 5'-ctcgagCCCTTTGCCCTTGCTcatatg -3';

[0248] Third step reaction template: SEQ ID No. 37: 5'-ctcgagAAACCCTTTGCCcatatg -3';

[0249] Fourth step reaction template: SEQ ID No. 33: 5'-ctcgagCCCAAACCCTTTGCCcatatg-3';

[0250] Fifth step reaction template: SEQ ID No. 39: 5'-ctcgacGGGCCCAAACCCgatatg -3';

[0251] Step 6 reaction template: SEQ ID No. 45: 5'-ctcgagTTTGGGCCCAAAcatatg-3';

[0252] Step 7 reaction template: SEQ ID No. 46: 5'-ctcgagCCCTTTGGGCCCAAAgatatg -3';

[0253] Step 8 reaction template: SEQ ID No. 47: 5'-ctcgagAAACCCTTTGGGcatatg -3';

[0254] Step 9 reaction template: SEQ ID No. 48: 5'-ctcgacTTTAAACCCTTTGGGgatatg -3';

[0255] Step 10 reaction template: SEQ ID No. 49: 5'-ctcgacGGGTTTAAACCCTTTgatatg -3'.

[0256] 3. The biotin-modified starter primer (SEQ ID No. 15) is bound to streptavidin magnetic beads, and the specific steps are the same as step 3 in Example 2.

[0257] 4. The first step, the enzymatic synthesis reaction, has the following specific steps:

[0258] a. Transfer the primers that have been bound to the magnetic beads into PCR tubes and add the reaction reagents. Each reaction is prepared according to the system shown in Table 9 below.

[0259] Table 9. First-step enzyme-catalyzed reaction system

[0260]

[0261] 10×Reaction Buffer: 400 mM Tris (pH 8.0), 60 mM KCL, 5 mM MgCL2, 10 mMDTT, 2.5% glycerol.

[0262] b. Place the PCR tube in the PCR instrument and react according to the procedure shown in Table 7;

[0263] c. After the reaction is complete, remove the PCR tube, place it on a magnetic rack for 1 minute to separate, and remove the supernatant;

[0264] d. Add 200 μL of washing buffer, thoroughly resuspend the magnetic beads by pipetting, and let stand for 2 min. Place the PCR tube on a magnetic rack for 1 min to separate, and remove the supernatant. Wash three more times with 1× reaction buffer before proceeding to the next step.

[0265] Washing buffer: 20 mM Tris (pH 8.0), 150 mM NaCl, 1 M sodium carbonate, 10% glycerol.

[0266] 5. Proceed to the second enzymatic synthesis reaction. The specific steps are the same as the first enzymatic synthesis reaction, except that the magnetic bead-starting primer in Table 9 is replaced with the product of the first reaction, the first reaction template SEQ ID No. 35 is replaced with the second reaction template SEQ ID No. 31, and dATP is replaced with dGTP.

[0267] 6. Proceed to steps three through ten of the synthesis.

[0268] 7. After the ten-step enzymatic reaction is completed, the final site extension is performed. The specific steps are as follows:

[0269] a. Design template for terminal point extension:

[0270] SEQ ID No. 50: 5'-CTCATGGTGGTGGTGGTGGTGCTCGAGGGTTTAAACCC-3'.

[0271] b. Prepare the reaction system as shown in Table 10;

[0272] Table 10 End-site extension reaction system

[0273]

[0274] c. Follow the procedure shown in Table 11;

[0275] Table 11 End-site extension reaction procedure

[0276]

[0277] d. After the extension reaction was completed, the enzymatically synthesized product was eluted from the magnetic beads with 8 M urea and purified using an oligonucleotide recovery kit (Sangon Biotech), following the kit's instructions. Finally, it was eluted with 20 μL of ultrapure water.

[0278] 8. Perform PCR and splice the product from step 7 (d) above. The specific steps are as follows:

[0279] a. Prepare the reaction systems as shown in Tables 12 and 3 respectively;

[0280] Table 12 Example 4 Fragment I PCR System

[0281]

[0282] b. Following the procedure shown in Table 4, PCR products of fragment I and fragment II were obtained respectively;

[0283] c. Perform a splicing reaction on fragment I and fragment II, prepare the reaction system as shown in Table 5, and carry out the reaction according to the procedure in Table 7 to obtain the spliced ​​product.

[0284] 9. Detection of enzyme-catalyzed synthesis products, the specific procedures are as follows:

[0285] The products from each enzymatic synthesis step were detected by 15% urea-polyacrylamide gel electrophoresis (voltage: 220 V, 1 h). After synthesis, Sanger sequencing and high-throughput sequencing were performed to analyze the synthesis efficiency and error rate.

[0286] 10. Results and Analysis

[0287] a. 15% urea-polyacrylamide gel electrophoresis detection

[0288] The 15% urea-polyacrylamide gel electrophoresis results of the enzymatically synthesized product in this embodiment are as follows: Figure 9 As shown, from left to right, these are the electrophoresis results of the synthetic products from steps one to ten, with the starting primer being 29-nt and one homotrimer added at each step. Figure 9 As can be seen, the template-dependent enzymatic synthesis method provided by the present invention can be continuously synthesized according to a specific nucleotide sequence, without base preference, with high synthesis efficiency, and can meet the needs of nucleic acid molecule synthesis.

[0289] b. Sanger sequencing and high-throughput sequencing detection of nucleic acid molecular variants

[0290] The Sanger sequencing and high-throughput sequencing results of the nucleic acid molecular variants obtained by enzymatic synthesis in this embodiment are as follows: Figure 10 As shown. By Figure 10 As can be seen, the present invention has a high synthesis accuracy, with an average single-step synthesis accuracy of 99.51%.

[0291] Example 5

[0292] Synthetic coding TDEDS ( T template d epended e nzymatic D NA s Nucleic acid molecular variants and storage accuracy assessment of ynthesis

[0293] 1. The DNA sequence encoding TDEDS

[0294] First, the ASCII codes corresponding to the five letters T, D, E, D, and S are each converted into five separate 5-bit ternary information entries. A 3-bit address code is added before each entry to indicate its alphabetical order. Then, this 8-bit ternary information is converted into five DNA sequences containing eight homotrimers of nucleotides, as shown in Table 13. Here, the first homotrimer (TTT in Table 13) is defined as 0 in the most significant (leftmost) bit of the ternary information. The remaining ternary information is represented using the homotrimer conversions, as shown in Table 14. In Table 14, A, T, C, and G represent AAA, TTT, CCC, and GGG, respectively; the conversion from TTT to CCC represents 0 in ternary; TTT to GGG represents 1; TTT to AAA represents 2, and so on, as shown in Table 14.

[0295] Table 13 Information Correspondence Results

[0296]

[0297] Table 14 Conversion Method Table

[0298]

[0299] 2. Construct and express the Dpo4 polymerase variant. The specific steps are the same as step 1 in Example 2. The difference is that this example only constructs and expresses Dpo4ΔC-CL7.

[0300] 3. Design and synthesize starting primers and templates.

[0301] The starting primer sequence is:

[0302] SEQ ID No. 15: 5'Biotin-GGAGAGCAGCT(6-FAM)ATGCAGCTTAGCAAGGGC-3'.

[0303] The template sequence is:

[0304] The template sequence required to synthesize a molecular variant encoding the first letter "T" is:

[0305] SEQ ID No.51: 5'-AAAGCCCTTGCTcatagtctc-3';

[0306] SEQ ID No.52: 5'-GGGAAAGCCCTTcatagtctc-3';

[0307] SEQ ID No.53: 5'-TTTGGGAAAGCCgatagtctc-3';

[0308] SEQ ID No.54: 5'-AAATTTGGGAAAcatagtctc-3';

[0309] SEQ ID No.55: 5'-GGGAAATTTGGGcatagtctc-3';

[0310] SEQ ID No.56: 5'-AAAGGGAAATTTcatagtctc-3';

[0311] SEQ ID No.57: 5'-CCCAAAGGGAAATTTcatagtctc-3';

[0312] SEQ ID No. 58:

[0313] 5'-CTCAGTGGTGGTGGTGGTGGTGCTCGATTTCCCAAAGGGcatagtctc-3'.

[0314] The template sequence required to synthesize a molecular variant encoding the second letter "D" is:

[0315] SEQ ID No.51: 5'-AAAGCCCTTGCTcatagtctc-3';

[0316] SEQ ID No.52: 5'-GGGAAAGCCCTTcatagtctc-3';

[0317] SEQ ID No.59: 5'-CCCGGGAAAGCCCTTcatagtctc-3';

[0318] SEQ ID No.60: 5'-TTTCCCGGGAAAcatagtctc-3';

[0319] SEQ ID No.61: 5'-GGGTTTCCCGGGcatagtctc-3';

[0320] SEQ ID No.62: 5'-TTTGGGTTTCCCgatagtctc-3';

[0321] SEQ ID No. 63: 5'-AAATTTGGGTTTCCCcatagtctc-3';

[0322] SEQ ID No. 64:

[0323] 5'-CTCAGTGGTGGTGGTGGTGGTGCTCGATTTAAATTTGGGTTTcatagtctc-3'.

[0324] The template sequence required to synthesize a molecular variant encoding the third letter "E" is:

[0325] SEQ ID No.51: 5'-AAAGCCCTTGCTcatagtctc-3';

[0326] SEQ ID No.65: 5'-CCCAAAGCCCTTGCTcatagtctc-3';

[0327] SEQ ID No.66: 5'-TTTCCCAAAGCCgatagtctc-3';

[0328] SEQ ID No.67: 5'-CCCTTTCCCAAAGCCgatagtctc-3';

[0329] SEQ ID No.68: 5'-AAACCCTTTCCCgatagtctc-3';

[0330] SEQ ID No.69: 5'-CCCAAACCCTTTCCCgatagtctc-3';

[0331] SEQ ID No.70: 5'-AAACCCAAACCCgatagtctc-3';

[0332] SEQ ID No. 71:

[0333] 5'-CTCAGTGGTGGTGGTGGTGGTGCTCGAGGGAAACCCAAAcatagtctc-3'.

[0334] The template sequence required to synthesize a molecular variant encoding the fourth letter "D" is:

[0335] SEQ ID No.51: 5'-AAAGCCCTTGCTcatagtctc-3';

[0336] SEQ ID No.65: 5'-CCCAAAGCCCTTGCTcatagtctc-3';

[0337] SEQ ID No.72: 5'-GGGCCCAAAGCCgatagtctc-3';

[0338] SEQ ID No.73: 5'-AAAGGGCCCAAAcatagtctc-3';

[0339] SEQ ID No.74: 5'-TTTAAAGGGCCCgatagtctc-3';

[0340] SEQ ID No.75: 5'-AAATTTAAAGGGcatagtctc-3';

[0341] SEQ ID No.76: 5'-CCCAAATTTAAAGGGcatagtctc-3';

[0342] SEQ ID No. 77:

[0343] 5'-CTCAGTGGTGGTGGTGGTGGTGCTCGAAAACCCAAATTTcatagtctc-3'.

[0344] The template sequence required to synthesize a molecular variant encoding the fifth letter "S" is:

[0345] SEQ ID No.51: 5'-AAAGCCCTTGCTcatagtctc-3';

[0346] SEQ ID No.65: 5'-CCCAAAGCCCTTGCTcatagtctc-3';

[0347] SEQ ID No.78: 5'-AAACCCAAAGCCgatagtctc-3';

[0348] SEQ ID No.79: 5'-CCCAAACCCAAAGCCgatagtctc-3';

[0349] SEQ ID No.80: 5'-TTTCCCAAACCCgatagtctc-3';

[0350] SEQ ID No.81: 5'-CCCTTTCCCAAACCCgatagtctc-3';

[0351] SEQ ID No.82: 5'-TTTCCCTTTCCCgatagtctc-3';

[0352] SEQ ID No. 83:

[0353] 5'-CTCATGGTGGTGGTGGTGGTGCTCGAGGGTTTCCCTTTcatagtctc-3'.

[0354] 4. Synthesize nucleic acid molecular variants encoding TDEDS

[0355] The specific steps in Example 4 were followed, including binding the biotin-modified starting primer to streptavidin magnetic beads, completing the eight-step synthesis of five sequenced nucleic acid molecular variants, extending the final site and purifying and recovering the product, and performing PCR and splicing on the recovered product.

[0356] 5. Detection of enzyme-catalyzed synthesis products

[0357] The products from each enzymatic synthesis step were detected by 15% urea-polyacrylamide gel electrophoresis (voltage: 220 V, 1 h). After synthesis, high-throughput sequencing was performed to analyze the synthesis and coding accuracy.

[0358] 6. Results and Analysis

[0359] a. 15% urea-polyacrylamide gel electrophoresis detection

[0360] The 15% urea-polyacrylamide gel electrophoresis results of the enzymatically synthesized product in this embodiment are as follows: Figure 11 As shown, from left to right, these are the electrophoresis results of the synthetic products from steps one to eight. The starting primer is 29-nt, and from top to bottom are the sequences of the nucleic acid molecular variants encoding T, D, E, D, and S. Figure 11As can be seen, the template-dependent enzymatic synthesis method provided by the present invention can be continuously synthesized according to a specific nucleotide sequence, with high synthesis efficiency, and can meet the needs of DNA data storage.

[0361] b. High-throughput sequencing detection of nucleic acid molecules

[0362] The high-throughput sequencing results of the nucleic acid molecular variants obtained by enzymatic synthesis in this embodiment are as follows: Figure 13 As shown, the synthesis and encoding accuracy are as follows: Figure 12 As shown, the average single-step synthesis accuracies for the synthesized nucleic acid molecular variants encoding T, D, E, D, and S were 98.75%, 98.78%, 99.38%, 99.47%, and 98.76%, respectively, while the coding accuracies were 100.00%, 100.00%, 99.89%, 100.00%, and 99.88%, respectively. Figure 13 and Figure 12 As can be seen, the present invention has high synthesis efficiency and high accuracy, and the coding accuracy is as high as 100.00%.

[0363] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0364] It should be noted that the above content merely illustrates the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. For those skilled in the art, various improvements and modifications can be made without departing from the principle of the present invention, and all such improvements and modifications fall within the scope of protection of the claims of the present invention.

Claims

1. A method for synthesizing a nucleic acid molecular variant, characterized in that, Includes the following steps: (1) Design and synthesize starting primers; (2) Fix the starting primers obtained in step (1) onto a solid support; (3) Add reaction buffer, target nucleotide molecule, template, and polymerase variant to the product obtained in step (2); (4) Denaturation-annealing-extension: Add the target nucleotide molecule to the 3' end of the primer; (5) Wash to obtain primers with one homopolymer added; (6) Repeat steps (3)-(5) until the sequence of the nucleic acid molecular variant is complete; (7) The product is peeled off from the solid support to obtain a nucleic acid molecular variant; The nucleic acid molecule variant is composed of nucleotide homopolymers, which are either homotrimers or homotetramers; In step (3), the polymerase variant is a Taq DNA polymerase variant or a Dpo4 polymerase variant; The Taq DNA polymerase variant is TaqΔN-TM, and the amino acid sequence of TaqΔN-TM is shown in SEQ ID No. 1; the Dpo4 polymerase variants include Dpo4ΔC and Dpo4ΔC-CL7, the amino acid sequence of Dpo4ΔC is shown in SEQ ID No. 2, and the amino acid sequence of Dpo4ΔC-CL7 is shown in SEQ ID No.

3.

2. The method for synthesizing a nucleic acid molecular variant according to claim 1, characterized in that, In step (3), the template is a single-stranded nucleic acid molecule variant, which consists of a core region and protective base portions at the 5' and 3' ends on both sides; the core region consists of two parts: a primer complementary portion and a guiding base portion; the primer complementary portion is complementary to the 3' end base of the primer; the guiding base portion guides the addition of nucleotides complementary to its bases; the primer complementary portion and the guiding base portion are each composed of nucleotide homopolymers.

3. The method for synthesizing a nucleic acid molecular variant according to claim 2, characterized in that, The core region has 21 or fewer nucleotides; the last nucleotide of the 5' protective base portion is different from the first nucleotide of the core region; the 3' protective base portion has 3 or more nucleotides.

4. The method for synthesizing a nucleic acid molecular variant according to claim 1, characterized in that, In step (4), the reaction conditions for denaturation-annealing-extension include: denaturation: 85-95℃, 5-15s; annealing: 30-50℃, 5-15s; extension: 50-65℃, 5-15s; 3-8 cycles.

5. The method for synthesizing a nucleic acid molecular variant according to claim 1, characterized in that, In step (5), the cleaning solution contains 0.5-2 M sodium carbonate.

6. The application of a nucleic acid molecular variant synthesized by the synthetic method according to any one of claims 1-5 in data storage.

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