A terminally modified deoxynucleotide

Abstract

The application discloses a terminal modified deoxynucleotide, and belongs to the technical field of DNA polymerization. The structural formula of the deoxynucleotide is as follows: wherein R is at least one of the following groups:,,, and. The terminal modified deoxynucleotide improves the stability of the deoxynucleotide in storage and in a PCR process, and the deoxynucleotide has the characteristics of heat instability, can be hydrolyzed to generate dNTP after being heated, can more long-term supplement raw materials required in the PCR process, and thus realizes amplification of a long DNA fragment, and has a wide application prospect.

Description

Technical Field

[0001] This application belongs to the field of deoxynucleotide technology, specifically relating to a terminal-modified deoxynucleotide. Background Technology

[0002] Polymerase chain reaction (PCR) is a fundamental technique in molecular biology research. Deoxynucleotide triphosphates (dNTPs) are essential raw materials for DNA synthesis, and their stability directly affects amplification efficiency. Currently, unmodified natural dNTPs are typically used directly in PCR reactions.

[0003] However, natural dNTPs have significant drawbacks in practical applications. On the one hand, natural dNTPs are prone to hydrolysis during storage, requiring stringent storage conditions to ensure their stability. On the other hand, during PCR reactions, due to the multiple high-temperature denaturation steps, natural dNTPs are also susceptible to hydrolysis and degradation under thermal cycling conditions, leading to a sharp decrease in the effective substrate concentration with increasing cycle number. Particularly in the amplification of long DNA fragments, insufficient dNTP supply in the later stages of the reaction causes premature termination of polymerase extension, making it difficult to obtain complete long-fragment products.

[0004] Therefore, how to solve the hydrolysis problem of natural dNTPs during storage and use, and achieve a stable supply of raw materials throughout the PCR process, especially in the later stages of the reaction, in order to meet the demand for efficient amplification of long DNA fragments, is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0005] In view of this, the primary objective of this application is to provide an end-modified deoxynucleotide that improves stability during storage and PCR, and is thermally unstable, capable of hydrolyzing to generate dNTPs upon heating, thus providing a longer-lasting supply of raw materials required for the PCR process and enabling the amplification of long DNA fragments.

[0006] To achieve the above objectives, this application adopts the following technical solution: One aspect of this application discloses a terminally modified deoxynucleotide, the structural formula of which is as follows: , Wherein, R is at least one of the following groups: , , , .

[0007] Another aspect of this application discloses the use of deoxynucleotides as described in this application in enzymatic DNA polymerization reactions.

[0008] Another aspect of this application discloses an enzymatic method for preparing nucleic acids, including: (a) the step of annealing the DNA primers and DNA template; and (b) the step of forming nucleic acids in the presence of DNA polymerase and the deoxynucleotides described in this application.

[0009] Another aspect of this application discloses a reagent combination for achieving nucleic acid extension, the reagent combination comprising: a DNA template, DNA primers, DNA polymerase, and the deoxynucleotides described in this application.

[0010] Another aspect of this application discloses a reagent kit containing the reagent combination described in this application.

[0011] The beneficial effects of this application are: This application discloses a thermally unstable terminal-modified deoxynucleotide that is stable during storage and PCR, but thermally unstable. When heated, it can be hydrolyzed to generate dNTPs, which can replenish the raw materials required for the PCR process for a longer period of time, thereby enabling the amplification of long DNA fragments. Attached Figure Description

[0012] Figure 1 The results of the thermal stability study of dNP4OC2H4NH2 in Example 2 are shown; where, Figure 1 (a) shows the HPLC results of dAP4OC2H4NH2 after heating at 90℃ for 20 min; Figure 1 (b) HPLC results of dAP4OC2H4NH2 after heating at 90℃ for 2 h; Figure 1 HPLC results of dAP4OC2H4NH2 after heating at 72℃ for 1 h (c); Figure 1 The result in (d) is the HPLC result of dAP4OC2H4NH2 after heating at 94℃ for 1 h.

[0013] Figure 2 The results of DNA polymerase recognition of dNP4OC2H4NH2 in Example 3 are shown; where, Figure 2 In the text, "+" indicates the addition of the compound, "-" indicates the absence of the compound, and "M" represents the DNA molecular weight standard marker (100-2000bp).

[0014] Figure 3 The results show the experimental results of the adaptability of dNP4OC2H4NH2 to different DNA polymerases in Example 4.

[0015] Figure 4 The results are from Example 5, which describes the application of dNP4OC2H4NH2 to the amplification of a 10kb long DNA fragment. Detailed Implementation

[0016] The embodiments of this application will be clearly and completely described below. The technical solutions in the embodiments described below are exemplary and only possible technical implementations of this application, not all possible implementations. Those skilled in the art can combine the embodiments of this application to obtain other embodiments without creative effort, and these embodiments are also within the protection scope of this application.

[0017] The first aspect of this application discloses a terminally modified deoxynucleotide, the structural formula of which is as follows: , Wherein, R is at least one of the following groups: , , , .

[0018] The preparation of the terminal-modified deoxynucleotide in this application can be carried out using techniques known in the art. Specifically, firstly, deoxynucleoside tetrabutylammonium triphosphate and O-phosphoethanolamine tetrabutylammonium salt are prepared; then, under suitable conditions, the deoxynucleoside tetrabutylammonium triphosphate and O-phosphoethanolamine tetrabutylammonium salt undergo a coupling reaction to obtain the terminal-modified deoxynucleotide.

[0019] As an example, the specific preparation steps of the terminal-modified deoxynucleotide in this application are as follows: <Preparation of Tetrabutylammonium Triphosphate of Deoxynucleoside> Sodium deoxynucleotides were converted to a free acid using a Dowex-50W ion exchange column (acidic form), then titrated to pH 7.0 with a dilute solution of tetrabutylammonium hydroxide. The solution was then concentrated to approximately one-seventh of its original volume by high-vacuum rotary evaporation and lyophilized. The lyophilized powder was dissolved in dry acetonitrile and dried. An equal volume of dry toluene was added, and the solution was concentrated to dryness by rotary evaporation, repeated three times. The residue was placed under high vacuum for 1 hour, then removed from the flask under argon protection, and dried DMF was added and filtered through a 4Å molecular sieve. After the solution was allowed to stand for at least 1 hour, it was used for subsequent coupling reactions.

[0020] As an example, the sodium deoxynucleotide used in this application is the disodium salt of deoxynucleoside triphosphate, and its specific structure is as follows: ; R is one of the bases A, G, T or C.

[0021] The structure of the prepared deoxynucleoside tetrabutylammonium triphosphate is shown below: .

[0022] <Preparation of O-phosphoethanolamine tetrabutylammonium salt> O-phosphoethanolamine was dissolved in deionized water, then diluted tetrabutylammonium hydroxide solution was added and the mixture was stirred until the pH reached 7.0. The solution was concentrated to approximately one-seventh of its original volume by high-vacuum rotary evaporation, followed by lyophilization. The lyophilized powder was dissolved in acetonitrile and dried, then an equal volume of dry toluene was added, and the mixture was concentrated to dryness by rotary evaporation, repeated three times. The residue was placed under high vacuum for 1 hour. Under argon protection, the residue was removed from the flask, dissolved in dry DMF, and 4 Å molecular sieve was added. This solution was allowed to stand for at least 1 hour before being used for subsequent coupling reactions.

[0023] In this application, the structure of O-phosphoethanolamine is as follows: .

[0024] The structure of the prepared O-phosphoethanolamine tetrabutylammonium salt is shown below:

[0025] <Preparation of terminal-modified deoxynucleotides> O-phosphoethanolamine tetrabutylammonium salt was dissolved in dry DMF, and CDI (N,N'-carbonyldiimidazole) was added. The mixture was stirred at room temperature for 12 hours, followed by quenching the reaction with methanol. All solvents were removed under high vacuum, and the residue was redissolved in anhydrous DMF. The desired deoxynucleoside triphosphate tetrabutylammonium salt was mixed with anhydrous DMF and anhydrous magnesium chloride. The mixture was stirred at room temperature for 72 hours. Then, RPHPLC purification was performed using a preparative C18 column through a gradient of acetonitrile and 50 mM triethylammonium acetate buffer (pH 7). The fraction containing the target product was collected, concentrated by high-vacuum rotary evaporation, and the residue was dissolved in water and repeatedly lyophilized until... 1 The 1H-NMR spectrum showed no residual buffer solution. The resulting white powder was converted to ammonium salt form using a Dowex-50W ion exchange resin.

[0026] The second aspect of this application discloses the use of the deoxynucleotides as described in claim 1 in enzymatic DNA polymerization reactions.

[0027] In this application, the "enzymatic DNA polymerization reaction" refers to the process by which, under the catalysis of DNA polymerase, deoxyribonucleoside triphosphates (dNTPs) are used as substrates, and nucleotides are added one by one to the 3'-OH end of a primer along the sequence of the DNA template strand, thereby synthesizing a new DNA strand complementary to the template strand. In this application, the substrate is specifically the terminal-modified deoxynucleotide described in this application.

[0028] It should be understood that enzymatic DNA polymerization encompasses a variety of specific applications, and the deoxynucleotides with terminal modifications described in this application are all applicable to these applications. Specific examples of these applications include DNA replication, DNA repair, cDNA synthesis, PCR reactions, rolling circle amplification, isothermal amplification, recombinant DNA, etc., but are not limited to these.

[0029] DNA replication refers to the process of amplifying the entire genome or fragments using double-stranded DNA as a template, either in vivo or in vitro, in a simulated environment.

[0030] DNA repair refers to the synthetic reactions involving DNA polymerases during the repair of DNA damage, which fill gaps or replace damaged nucleotides.

[0031] cDNA synthesis is the process of synthesizing the first strand of complementary DNA (cDNA) using RNA as a template under the catalysis of reverse transcriptase, followed by the synthesis of the second strand by DNA polymerase.

[0032] Polymerase chain reaction (PCR) is an in vitro enzymatic amplification method for specific DNA fragments. It achieves exponential amplification of target DNA through three temperature cycles: denaturation, annealing, and extension.

[0033] Rolling circle amplification (RCA) is an isothermal amplification method that uses circular DNA as a template and generates tandem DNA containing a large number of repetitive sequences through primer binding and continuous extension by DNA polymerase.

[0034] Isothermal amplification refers to nucleic acid amplification reactions performed at a constant temperature, such as loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), and strand displacement amplification (SDA). These methods typically utilize DNA polymerases with strand displacement activity.

[0035] Recombinant DNA technology includes, but is not limited to, DNA fragment completion reactions before ligation in molecular cloning, site-directed mutagenesis, and DNA sequencing reactions.

[0036] It should be understood that any application in the art that covers enzymatic DNA polymerization reactions, including the deoxynucleotides modified at the ends in this application, is applicable and is not limited to the examples described above.

[0037] The third aspect of this application discloses an enzymatic method for preparing nucleic acids, characterized by comprising: (a) the step of annealing the DNA primers and DNA template; and (b) the step of forming nucleic acids in the presence of DNA polymerase and the deoxynucleotides as described in claim 1.

[0038] The fourth aspect of this application discloses a reagent combination for achieving nucleic acid extension, the reagent combination comprising: a DNA template, DNA primers, DNA polymerase, and the deoxynucleotides as described in claim 1.

[0039] Furthermore, the reagent combination also includes a buffer solution compatible with the DNA polymerase.

[0040] The fifth aspect of this application discloses a reagent kit containing the reagent combination described in this application.

[0041] In this application, the term "DNA primer" refers to a short, single-stranded oligodeoxyribonucleotide sequence. It is complementary to a specific region of the DNA template strand and can hybridize with the template strand under suitable conditions (such as appropriate temperature and buffer system) to form a local double-stranded structure. The 3' end of the DNA primer contains a free hydroxyl group (—OH), which is the starting point for DNA polymerase to initiate the nucleotide polymerization reaction. Specifically, the length, sequence, and modification of the DNA primer can be adjusted or selected according to specific experimental purposes or research needs. Specific examples include, but are not limited to: universal primers, such as M13 forward / reverse primers, T7 primers, SP6 primers, etc., which are complementary to the vector sequence; specific primers, oligonucleotides designed for target genes or specific sequences; random primers, mixtures of oligonucleotides composed of random sequences, such as random hexamer primers, which are often used for cDNA synthesis or whole genome amplification; and modified primers, including but not limited to primers with biotin, fluorescent groups (such as FAM, Cy, etc.), phosphate groups introduced at the 5' end, or primers with phosphorylation, aminoation modifications at the 3' end, or primers with the introduction of phosphate thioester bonds to enhance nuclease resistance.

[0042] In this application, the term "DNA template" refers to a nucleic acid molecule that serves as a guide for the synthesis of a complementary DNA strand. In an enzymatic DNA polymerization reaction, DNA polymerase reads the base sequence of the template strand and adds the corresponding deoxyribonucleotides to the primer ends according to the base pairing principle (A pairs with T, G pairs with C). It is understood that DNA templates come in various sources and forms, and those skilled in the art can select them according to experimental purposes and research needs. Specific examples can be genomic DNA extracted from various biological samples (such as blood, tissues, cells, and microorganisms); plasmid DNA, i.e., recombinant or natural circular double-stranded DNA molecules; PCR amplification products, serving as templates for the next round of amplification (such as nested PCR) or sequencing reactions; or cDNA, complementary DNA produced by reverse transcription of mRNA. Furthermore, single-stranded DNA, such as single-stranded DNA produced by asymmetric PCR or bacteriophages, or chemically synthesized oligonucleotides, can be used as templates for the synthesis of specific short fragments.

[0043] In this application, the term "DNA polymerase" refers to a class of enzymes capable of catalyzing the polymerization of deoxyribonucleotides to form DNA chains. Its primary activities include 5'→3' DNA polymerase activity, i.e., the ability to extend the DNA chain along the 5'→3' direction. Different DNA polymerases may possess one or more of the following auxiliary activities. Specific examples include, but are not limited to: 3'→5' exonuclease activity (proofreading activity): This activity enables the recognition and excision of mismatched nucleotides, improving the fidelity of DNA synthesis. For example, DNA polymerase from Pyrococcus furiosus (Pfu) possesses this activity.

[0044] 5'→3' exonuclease activity: This activity involves the excision of DNA or RNA fragments located ahead of the synthetic strand, playing a role in nick translation or TaqMan probe hydrolysis. For example, the DNA polymerase from Thermus aquaticus (Taq) possesses this activity.

[0045] Strand substitution activity: This refers to the ability to replace the complementary strand of downstream DNA during synthesis without degrading it. Examples include phi29 DNA polymerase and large fragments of Bst DNA polymerase, which are key enzymes in rolling circle amplification and isothermal amplification.

[0046] The specific type of DNA polymerase can be selected according to the experimental purpose and research needs, and those skilled in the art are capable of doing so.

[0047] It should be understood that different DNA polymerases require different buffer solutions. These buffer solutions are solution systems that provide the optimal chemical environment for enzymatic DNA polymerization reactions. They maintain the pH stability of the reaction system and provide the cofactors and auxiliary factors necessary for DNA polymerase activity.

[0048] The composition and concentration of these buffer solutions can be adjusted or selected according to the specific type of DNA polymerase and the experimental purpose. However, the specific composition typically includes the following components: Buffering components: Used to maintain pH stability, typically Tris-HCl (tris(hydroxymethyl)aminomethane) hydrochloride, whose pH value (usually between 7.5 and 9.0, e.g., 7.5, 8.0, 8.5, 9.0) can be selected according to the optimal pH of the polymerase.

[0049] Monovalent cation salts, such as KCl (potassium chloride), NaCl (sodium chloride), and NH4Cl (ammonium chloride), can be available in concentrations ranging from 10mM to 100mM, and are used to provide ionic strength and stabilize the double-stranded structure of DNA.

[0050] Divalent cations: such as Mg2+ (Usually provided in the form of MgCl2 or MgSO4), it is an essential cofactor for almost all DNA polymerases, and its concentration is crucial to enzyme activity and specificity. Common concentration ranges include, for example, 1.0 mM, 1.5 mM, 2.0 mM, 2.5 mM, 3.0 mM, 4.0 mM, 5.0 mM, etc.

[0051] Stabilizers and enhancers include, but are not limited to, bovine serum albumin (BSA), gelatin, nonionic detergents (such as Triton X-100, Tween 20), dimethyl sulfoxide (DMSO), betaine, etc., used to improve enzyme stability and amplification efficiency.

[0052] Reducing agents, such as dithiothreitol (DTT), are used to maintain the active conformation of enzymes.

[0053] The above are merely examples of buffer solutions. It is understood that appropriate adjustments are permissible depending on the specific circumstances, which will not be elaborated upon here.

[0054] In this application, the term "kit" refers to a product form in which relevant components are brought together to perform an enzymatic DNA polymerization reaction. This kit pre-packages and assembles the core components required for the reaction to simplify the process and ensure the stability and reproducibility of the reaction results.

[0055] The kit of this application contains at least the terminal-modified deoxynucleotides described in this application, and may further contain DNA polymerase and a matching buffer solution, DNA primers, and DNA templates (such as standards or controls). Preferably, it contains all of the above-mentioned reagents. Furthermore, depending on the application, such as a PCR premix kit, cDNA synthesis kit, isothermal amplification kit, or DNA sequencing kit, it may further contain other auxiliary reagents adapted to the above applications, such as nucleic acid dyes, loading buffers, and purification columns.

[0056] The present application will be further illustrated below with reference to specific embodiments. It should be noted that the specific embodiments below are for illustrative purposes only and do not limit the scope of the present application in any way.

[0057] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the specification of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application.

[0058] In addition, unless otherwise specified, methods without detailed conditions or steps are conventional methods, and the reagents and materials used are commercially available.

[0059] Example 1: Terminally Modified Deoxynucleotides and Their Preparation 1.1 Terminally modified deoxynucleotides Its specific structure is as follows: ; Wherein, R is at least one of the following groups: , , , .

[0060] 1.2 Preparation of terminally modified deoxynucleotides (1) Preparation of deoxynucleoside tetrabutylammonium triphosphate Disodium deoxyribonucleotide (515 mg) was converted to a free acid using a Dowex-50W ion exchange column (5 g) (acid form), then titrated to pH 7.0 with an aqueous solution of tetrabutylammonium hydroxide (25% by mass). The solution was then concentrated to approximately one-seventh of its original volume by rotary evaporation under high vacuum (-0.098 MPa) and lyophilized. The lyophilized powder was dissolved in dry acetonitrile and dried. An equal volume of dry toluene was added, and the solution was concentrated to dryness by rotary evaporation, repeated three times. The residue was placed under high vacuum for 1 hour, then removed from the flask under argon protection, dried DMF was added, and the mixture was filtered through a 4 Å molecular sieve. After standing for at least 1 hour, the solution was used for subsequent coupling reactions.

[0061] (2) Preparation of O-phosphoethanolamine tetrabutylammonium salt O-phosphoethanolamine (705 mg) was dissolved in deionized water (1 mL), followed by the addition of an aqueous solution of tetrabutylammonium hydroxide (25% by mass) and stirring until the pH reached 7.0. The solution was concentrated to approximately one-seventh of its original volume by high-vacuum rotary evaporation, and then lyophilized. The lyophilized powder was dissolved in acetonitrile and dried, and an equal volume of dry toluene was added. The solution was then concentrated to dryness by rotary evaporation, repeated three times. The residue was placed under high vacuum for 1 hour. Under argon protection, the solution was removed from the flask, dissolved in dry DMF, and 4 Å molecular sieve was added. This solution was allowed to stand for at least 1 hour before being used for subsequent coupling reactions.

[0062] (3) Synthesis of terminally modified deoxynucleotides Dissolve 25.5 mg, 40 μmol, 1.0 equivalent of O-phosphoethanolamine tetrabutylammonium salt in 1.0 mL of dry DMF, add CDI (N,N'-carbonyldiimidazole) (32.3 mg, 200 μmol, 5.0 equivalent), and stir the mixture at room temperature for 12 hours. Then, quench the reaction with 50 μL of methanol. All solvents were removed under high vacuum, and the residue was redissolved in 1 mL of anhydrous DMF. The desired deoxynucleoside triphosphate tetrabutylammonium salt (40 μmol) was mixed with 1 mL of anhydrous DMF containing the residue and anhydrous magnesium chloride (5 mg). The mixture was stirred at room temperature for 72 hours. Then, RPHPLC purification was performed using a preparative C18 column via a gradient of acetonitrile and 50 mM triethylammonium acetate buffer (pH 7). The fraction containing the target product was collected, concentrated by high-vacuum rotary evaporation, and the residue was dissolved in water and repeatedly lyophilized until... 1 The 1H-NMR spectrum showed no residual buffer solution. The resulting white powder was converted to ammonium salt form using a Dowex-50W ion exchange resin.

[0063] dAP4OC2H4NH2: 1 H NMR (D2O, 600 MHz) δ 8.34 (s, 1H), 8.09 (s, 1H), 6.34 (dt, J = 23.7, 6.9 Hz, 1H), 4.15 (s, 1H), 4.07 (s, 4H), 3.46 (s, 1H), 3.12(s, 1H), 2.67 (s, 1H), 2.42 (s, 1H); 31 P NMR (D2O, 243 MHz) δ 10.93 - 11.74 (m), 22.81 - 23.68 (m); HRMS (ESI) calcd for C 12 H 22 N6O 15 P4[MH] - 613.0021, found 613.0033. dTP4OC2H4NH2: 1 H NMR (D2O, 600 MHz) δ 7.57 (s, 1H), 6.17 (dt, J = 20.1,7.0 Hz, 1H), 4.51 (s, 1H), 4.13 (s, 1H), 4.07 (s, 4H) 3.50 (s, 1H), 3.10 (s,1H), 2.19 (s, 2H);31 P NMR (D2O, 243 MHz) δ 10.94 - 11.78 (m), 23.12 - 23.39(m); HRMS (ESI) calcd for C 12 H 23 N3O 17 P4[M-H] - 603.9905, found 603.9884. dCP4OC2H4NH2: 1 H NMR (D2O, 600 MHz) δ 7.80 (s, 1H), 6.14 (dt, J = 25.2,6.7 Hz, 1H), 5.96 (dd, J = 17.4, 7.6 Hz, 1H), 4.44 (s, 1H), 4.05 (s, 5H),3.50 (s, 1H), 3.13 (s, 1H), 2.16 (s, 2H); 31 P NMR (D2O, 243 MHz) δ 11.07 - 11.22 (m), 23.04 - 23.18 (m); HRMS (ESI) calcd for C 11 H 22 N6O 16 P4[M-H] - 588.9909,found 589.0528. dGP4OC2H4NH2: 1 H NMR (D2O, 600 MHz) δ 8.00 (s, 1H), 6.16 (dt, J = 18.8,7.0 Hz, 1H), 4.66 (s, 1H), 4.10 (s, 5H), 3.53 (s, 1H), 3.16 (s, 1H), 2.70 (s,1H), 2.38 (s, 1H); 31 P NMR (D2O, 243 MHz) δ 11.45 - 11.68 (m), 23.34 - 23.53(m); HRMS (ESI) calcd for C 12 H 22 N6O 16 P4[MH] - 628.9970, found 629.0633. Example 2: Thermal stability study of dNP4OC2H4NH2 5 μL of dAP4OC2H4NH2 (200 ng / μL) was dissolved in 20 μL of Taq DNAase buffer. The solutions were heated at 90 °C for 20 min and 180 min, respectively. The decomposition of the compounds was analyzed by HPLC. The results are as follows: Figure 1 As shown in the image.

[0064] It can be seen that after heating at 90℃ for 20 min, dAP4OC2H4NH2 was completely converted into the more stable dAP4OC2H4NH2 internal salt form. Small amounts of dATP were generated after heating at 90℃ for 2 h and at 94℃ for 1 h, while only trace amounts of dATP were generated after heating at 72℃ for 1 h. Figure 1 This indicates that dNP4OC2H4NH2 can serve as a thermally unstable nucleotide with an end label, which can be hydrolyzed upon heating to generate dNTPs, thus replenishing the raw materials during the PCR process.

[0065] Example 3: DNA polymerase recognition of dNP4OC2H4NH2 Perform the PCR reaction according to the PCR reaction system and procedure in Table 1.

[0066] Table 1 PCR reaction system and reaction procedure

[0067] Note: In Table 1, PET-28a is a universal and efficient prokaryotic expression vector based on the T7 promoter. The PET-28a-FMO plasmid is a recombinant vector formed by inserting the FMO (flavin monooxygenase) gene into the universal vector PET-28a. It can be obtained by a third party.

[0068] PCR products were analyzed by gel electrophoresis on a 0.8% agarose gel containing Nucleic Acid Stain. The gel was placed in 1×Tris-Borate-EDTA (TBE) buffer and run at 150V for 0.5 h. The gels were visualized using a GenoSens 2000 gel imaging system, and band intensity was analyzed using ImageJ software (National Institutes of Health). A control group with 200 μM dNTPs was also included. Results Figure 2 As shown.

[0069] The primer information is shown in Table 2: Table 2 Primer Information

[0070] Figure 2 The results showed that the dNP4OC2H4NH2 in Example 1 could be successfully used for 1kb DNA amplification, indicating that the terminal-modified dNP4OC2H4NH2 nucleotide analogues can be applied to PCR amplification technology.

[0071] Example 4: Adaptability of dNP4OC2H4NH2 to different DNA polymerases In this embodiment, a 1kb fragment is still used as a template for PCR amplification. The PCR reaction system and procedure are shown in Table 1, and primer information is shown in Table 2. The difference lies in the use of different DNA polymerases: Taq, Pfu, or LongAmp® Taq DNA polymerase for PCR amplification. It is understood that when using different DNA polymerases, the buffer solution is replaced with the corresponding 1× buffer solution.

[0072] The electrophoresis and band observation methods were the same as in Example 3. The results are as follows: Figure 3 As shown in the figure, using Taq, Pfu, and LongAmp® Taq DNA polymerase, and performing PCR amplification experiments using dNP4OC2H4NH2, all successfully amplified 1kb DNA fragments.

[0073] Example 5: dNP4OC2H4NH2 applied to 10kb long DNA fragment amplification Perform the PCR reaction according to the PCR reaction system and procedure in Table 3.

[0074] Table 3 PCR reaction system and reaction procedure

[0075] The electrophoresis and band observation methods were the same as in Example 3. A control group with 200 μM of natural dNTPs was also set up. Experimental Results Figure 4 As shown.

[0076] The primer information is shown in Table 4: Table 4 Primer Information

[0077] Figure 4In the diagram, 1 represents dNTPs heated at 72℃ for 12 hours; 2 represents dNP4OC2H4NH2 heated at 72℃ for 12 hours; 3 represents dNTPs after 80 freeze-thaw cycles; 4 represents dNP4OC2H4NH2 after 80 freeze-thaw cycles; and M represents λDNA-HindIII enzymatic digestion products. Figure 4 The results show that for amplifying long DNA fragments of 10kb, the dNP4OC2H4NH2 in this application can effectively resist high temperatures, while the natural dNTPs failed to amplify. The terminal-modified deoxynucleotides in this application have a wider range of application prospects.

[0078] It should be noted that this application is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments with the same structure and effect as the technical concept within the scope of this application are included in the technical scope of this application. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of this application, are also included in the scope of this application.

Claims

1. A terminally modified deoxynucleotide, characterized in that, The structural formula of the deoxynucleotide is as follows: ， Wherein, R is at least one of the following groups: 、 、 、 。 2. The application of the deoxynucleotide as described in claim 1 in enzymatic DNA polymerization.

3. Use according to claim 2, wherein the compound is ###0002### The substrate in the enzymatic DNA polymerization reaction is the deoxynucleotide described in claim 1.

4. Enzymatic method for the preparation of nucleic acids, characterized in that, include: (a) The step of annealing the DNA primers and DNA template; (b) the step of forming nucleic acids in the presence of DNA polymerase and the deoxynucleotides as described in claim 1.

5. The enzymatic method of claim 4, wherein, The method is one of DNA replication, DNA repair, cDNA synthesis, PCR reaction, rolling circle amplification, isothermal amplification, or recombinant DNA.

6. A reagent combination for effecting nucleic acid elongation, characterized in that, The reagent combination includes: a DNA template, DNA primers, DNA polymerase, and the deoxynucleotides as described in claim 1.

7. The agent combination according to claim 6, wherein The reagent combination also includes a buffer solution compatible with the DNA polymerase.

8. A reagent kit, characterized in that, The kit contains the reagent combination as described in claim 6 or 7.

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