DNA polymerase, and preparation method therefor and use thereof

Abstract

Provided are a DNA polymerase, and a preparation method therefor and the use thereof. The DNA polymerase has less than 42% sequence similarity to Taq DNA polymerase, and has polymerase activity, higher thermal stability, 5'-3' exonuclease activity and inhibition resistance.

Description

DNA polymerase and its preparation method and application Technical Field

[0001] The present application belongs to the field of biotechnology and relates to DNA polymerase and its preparation method and application. Background Art

[0002] Nucleic acid amplification is a crucial technology in biological research, medical testing, forensic science, agriculture, and other related fields. Currently, the most widely used nucleic acid amplification technology is the polymerase chain reaction (PCR), which relies on DNA polymerase. DNA polymerases have multiple functions and are extremely important tool enzymes in molecular biology. For example, Taq DNA polymerase, which has high industrial value, is widely used in molecular diagnostics. Taq DNA polymerase belongs to the A family of DNA polymerases. Other members of this family include Tth DNA polymerase, which has reverse transcriptase activity, Bst DNA polymerase, which has strand displacement activity, and bacteriophage T7 DNA polymerase, which can bind 2',3'-dideoxynucleotides.

[0003] A suitable DNA polymerase is essential for a successful PCR reaction. Different DNA polymerases can be selected based on the sensitivity, fidelity, fragment length, and other reaction conditions required for each experiment. Furthermore, A-family DNA polymerases primarily possess 5'-3' polymerization and 5'-3' exolytic activities, making them widely used in molecular diagnostics, particularly Taq DNA polymerase.

[0004] Currently, the most widely used DNA polymerase is Taq DNA polymerase, a member of the A family. The discovery and development of Taq DNA polymerase spans nearly 50 years. In recent years, due to its crucial role in molecular diagnostics, the enzyme has been the subject of extensive research on its modification, and many commercial enzymes have been developed based on it. However, with the increasing diversification of application scenarios, new performance requirements for the enzyme have been continuously raised. Traditional directed evolution methods alone have limited scope for modification and are unable to meet the growing demand.

[0005] In summary, in order to develop new molecular tools with a wider range of applications, it is still necessary to explore new A family DNA polymerase backbones.

[0006] Summary of the Invention

[0007] This application provides DNA polymerase and its preparation method and application, and explores new DNA polymerases.

[0008] In a first aspect, the present application provides a DNA polymerase, wherein the amino acid sequence of the DNA polymerase comprises:

[0009] (1) the sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2; or

[0010] (2) an amino acid sequence obtained by substituting, deleting or adding one or at least two amino acid residues from the sequence described in (1), and having the same or similar function as the sequence described in (1); or

[0011] (3) An amino acid sequence that has at least 90% sequence homology with the sequence described in (1) or (2) and has the same or similar functions as the sequence described in (1).

[0012] In this application, based on the analysis of metagenomic sequencing data of deep-sea hydrothermal sediment samples, two protein sequences with the functions of A family DNA polymerases were discovered (named 34°S-1 and 34°S-2). After sequence alignment, the sequence similarities with Taq DNA polymerase were only 40.92% and 41.94%, respectively, which are completely new sequence skeletons, indicating that these two proteins can be used as new DNA polymerases.

[0013] It can be understood that the present application discovered that the two proteins with amino acid sequences such as those shown in SEQ ID NO.1 or SEQ ID NO.2 have the function of A family DNA polymerase. Therefore, the enzyme obtained by using the general technical means in the art to replace, delete or add one or at least two amino acid residues in the sequence shown in SEQ ID NO.1 or SEQ ID NO.2, and at the same time having the same or similar functions as the original protein, can also be expected to have the function of A family DNA polymerase.

[0014] In a second aspect, the present application provides a nucleic acid molecule encoding the DNA polymerase described in the first aspect.

[0015] Preferably, the nucleic acid sequence of the nucleic acid molecule includes the sequence shown in SEQ ID NO.3 or SEQ ID NO.4.

[0016] In a third aspect, the present application provides a recombinant vector comprising the nucleic acid molecule described in the second aspect.

[0017] In a fourth aspect, the present application provides a recombinant cell, wherein the recombinant cell contains the recombinant vector described in the third aspect.

[0018] In a fifth aspect, the present application provides a method for preparing the DNA polymerase described in the first aspect, the preparation method comprising:

[0019] The coding gene of the DNA polymerase described in the first aspect is inserted into an expression vector to obtain a recombinant vector, the recombinant vector is introduced into a host cell to obtain a recombinant cell, the recombinant cell is cultured, and the cultured cells are collected for product purification to obtain the DNA polymerase.

[0020] Preferably, the expression vector comprises a pET28a vector.

[0021] Preferably, the host cell comprises Escherichia coli (E. coli) BL21.

[0022] Preferably, the method for purifying the product comprises purifying using a nickel column-ion column.

[0023] In a sixth aspect, the present application provides the use of the DNA polymerase described in the first aspect in the preparation of a nucleic acid amplification product.

[0024] In a seventh aspect, the present application provides the use of the nucleic acid molecule described in the second aspect, the recombinant vector described in the third aspect, or the recombinant cell described in the fourth aspect in the preparation of DNA polymerase.

[0025] In an eighth aspect, the present application provides a nucleic acid amplification kit, which includes the DNA polymerase described in the first aspect.

[0026] In a ninth aspect, the present application provides the use of the DNA polymerase described in the first aspect in nucleic acid amplification.

[0027] In this application, the new DNA polymerase discovered has the functions of A family DNA polymerase and can be widely used in nucleic acid amplification, such as PCR amplification sequencing, library construction, qPCR, TA cloning and other applications.

[0028] In a tenth aspect, the present application provides a method for nucleic acid amplification, comprising: mixing a nucleic acid template with a reaction system containing the DNA polymerase described in the first aspect, and performing an amplification reaction.

[0029] Compared with the prior art, this application has the following beneficial effects:

[0030] This application discovered a new type of DNA polymerase with 5'-3' polymerization activity and 5'-3' exoclease activity, which is consistent with the functional activity characteristics of A family DNA polymerases. The sequence similarity with Taq DNA polymerase is only 40.92% and 41.94%. It is a brand-new sequence skeleton with greater room for product transformation and provides a new polymerase skeleton for the development of new molecular tools with a wider range of applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 shows the results of purification of 34°S-1 DNA polymerase.

[0032] Figure 2 shows the results of 34° S-2 DNA polymerase purification.

[0033] FIG3 is a graph showing the Tm values ​​of 34°S-1, 34°S-2 DNA polymerase, and Taq DNA polymerase.

[0034] FIG4 is a schematic diagram showing the principle of determining the polymerization activity of DNA polymerase.

[0035] FIG5 is a schematic diagram showing the principle of determining the 5′-3′ exonuclease activity of DNA polymerase.

[0036] FIG6 is a graph showing the 5'-3' exo-cleavage activity of 34°S-1, 34°S-2 DNA polymerases, and Taq DNA polymerase.

[0037] FIG7 is a diagram showing the results of PCR amplification using 34° S-1 DNA polymerase.

[0038] FIG8 is a graph showing the results of PCR amplification using 34° S-2 DNA polymerase.

[0039] FIG9 is a graph showing the results of 34° S-2 DNA polymerase inhibition resistance assay. DETAILED DESCRIPTION

[0040] To further illustrate the technical means and effects of this application, the following further describes this application in conjunction with examples and drawings. It should be understood that the specific implementation methods described herein are only used to explain this application, rather than to limit this application.

[0041] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0042] Example 1

[0043] This example performs sequence alignment.

[0044] The Clustal Omega online sequence alignment website was used to compare the sequences of the novel DNA polymerases 34°S-1 (amino acid sequence SEQ ID NO.1) and 34°S-2 (amino acid sequence SEQ ID NO.2) with the existing Taq DNA polymerase (GenBank: AYJ71526.1). The comparison results showed that the sequence identities of 34°S-1 polymerase and 34°S-2 polymerase with Taq DNA polymerase were 40.92% and 41.94%, respectively. The sequence similarity between the novel DNA polymerases 34°S-1 and 34°S-2 was 56.84%, indicating that a completely new sequence framework has been discovered in this application, and there is greater room for product transformation.

[0045] Example 2

[0046] This example constructs a recombinant plasmid and expresses and purifies it.

[0047] Changzhou Xinyisheng Biotechnology Co., Ltd. was commissioned to synthesize the gene sequences of 34°S-1 polymerase and 34°S-2 polymerase (SEQ ID NO.3 and SEQ ID NO.4), and the genes were cloned into the pET28a expression vector with Nde I and Xho I cloning sites. The recombinant plasmids were transformed into Escherichia coli BL21 (DE3) competent cells, and the plates were incubated at 37°C overnight for subsequent expression purification.

[0048] The affinity chromatography column used for protein purification is HisTrap FF 5mL. The specific protein expression and purification steps are as follows:

[0049] (1) Pick 5 single colonies that grow well on the plate and inoculate them into a 50 / 250 mL LB liquid conical flask. Incubate at 37°C for 6 h. 600 Then, the above bacterial solution was inoculated into 2L / 5L LB medium at a 1% inoculum volume and cultured at 37℃ for 3h. 600 Reach 0.8-1.0; pre-cool the original shaker to 16°C, add IPTG to the culture medium to a final concentration of 0.5mM; shake at 16°C, 220rpm, and induce expression for 14h;

[0050] (2) The cells were collected by centrifugation at 8000 g for 30 min, and then resuspended in Ni column affinity buffer (Ni column-A buffer) at a ratio of 1:20. The cells were then disrupted by ultrasound in an ice bath.

[0051] (3) The ultrasonicated solution was centrifuged at 12000 rpm for 60 min at 4°C, and the supernatant was filtered through a 0.22 μM filter membrane to serve as the sample for the purification column;

[0052] (4) The sample was loaded onto the pretreated chromatographic column (HisTrap FF 5 mL) at a rate of 3 mL / min. After loading, the column was rinsed with Ni column affinity solution A (Ni column-A Buffer) for 20 CV. Then, a linear elution of 10.5 CV was performed with Ni column affinity solution B (Ni column-B Buffer) at a ratio of 0-70%. The eluted protein was collected when the UV absorption peak reached 50 mAu, and the collection was stopped when the UV absorption peak dropped to 100 mAu.

[0053] (5) The collected eluate was diluted 9-fold with diluent and then loaded onto a pretreated Q column (HiTrap Q HP 5 mL). After loading, the column was rinsed with Q column A buffer for 10 CV until the baseline was stable at a flow rate of 5 mL / min. The target protein was gradient eluted with Q column B buffer (0-100% Q column B buffer, 10 CV) at a flow rate of 5 mL / min. The collected samples were analyzed by SDS-PAGE to determine the protein purity.

[0054] The purified protein sample was dialyzed and its concentration was determined, then stored in enzyme storage solution for subsequent functional activity analysis.

[0055] The specific components of the buffer used in the purification process are as follows:

[0056] Ni column-A Buffer: 20mM Tris-HCl, 300mM NaCl, 20mM Imidazole, 5% Glycerol, pH 7.8;

[0057] Ni column-B Buffer: 20mM Tris-HCl, 300mM NaCl, 500mM Imidazole, 5% Glycerol, pH 7.8;

[0058] Q column-A Buffer: 20mM Tris-HCl, 50mM NaCl, 5% Glycerol, pH 8.0;

[0059] Q column-B Buffer: 20mM Tris-HCl, 1M NaCl, 5% Glycerol, pH 8.0;

[0060] Diluent: 20 mM Tris-HCl, 5% Glycerol, pH 8.0;

[0061] 2× dialysate: 40 mM Tris-HCl, 200 mM KCl, 2 mM DTT, 0.2 mM EDTA, 5% Glycerol, pH 8.0;

[0062] Enzyme stock solution: 10 mM Tris-HCl, 100 mM KCl, 1 mM DTT, 0.1 mM EDTA, 50% Glycerol, Tween-20 0.5%, NP-40 0.5%, pH 8.0.

[0063] The purification results are shown in Figures 1 and 2, and the target protein of the expected size was obtained.

[0064] Example 3

[0065] In this example, the thermal stability of a novel DNA polymerase was determined.

[0066] Using Protein Thermal Shift TM The protein stability was determined using a dye kit (ThermoFisher) according to the kit instructions. Taq DNA polymerase was used as a control. The results are shown in Figure 3. It can be seen that the Tm values ​​of 34°S-1 and 34°S-2 DNA polymerases and Taq DNA polymerase (Sangon Biotechnology, Product No. B600001) are 95.3°C, 96.8°C, and 93.2°C, respectively, indicating that the new polymerase discovered in this application has higher thermal stability.

[0067] Example 4

[0068] In this example, polymerization activity was measured.

[0069] Polymerization activity was determined using a primed M13 ssDNA substrate. The specific principle is shown in Figure 4 . In the presence of polymerization activity, the primers on the primed M13 ssDNA will extend along the ssDNA in the 5'-3' direction to produce dsDNA, which can be quantitatively detected using the Qubit dsDNA HS Assay Kits (ThermoFisher). The specific reaction system and components are shown in Table 1.

[0070] Table 1

[0071] The NC group did not add polymerase and was filled with water. The reaction mixture was incubated at 72°C for 5 minutes, then 1 μL of 0.5 M EDTA was added to terminate the reaction. The dsDNA concentration was determined using the Qubit dsDNA HS Assay Kit according to the manufacturer's instructions. The results are shown in Table 2, indicating that 34°S-1 and 34°S-2 DNA polymerases exhibit polymerization activity at 72°C.

[0072] Table 2

[0073] Example 5

[0074] In this example, exo-cleavage activity assay (5'-3' exo-cleavage activity) was performed.

[0075] For the detection of exo-cleavage activity, the Taqman probe method was used. When the 5'-3' exo-cleavage activity was activated, the downstream probe chain was degraded by transferring the activity through the incision (as shown in Figure 5). The generated fluorescent signal was detected by a microplate reader, and the 5'-3' exo-cleavage determination was performed based on the change in the signal.

[0076] Annealing of fluorescent probe substrate: Place the prepared system into a PCR instrument and react at 90°C for 2 minutes, then turn off the power and allow to cool naturally.

[0077] The reaction system is shown in Table 3 below.

[0078] Table 3

[0079] Detection and fluorescence signal collection were performed using a microplate reader, with excitation at 494 nm and absorption at 522 nm. Specifically, the reaction was allowed to proceed at 37°C for 1 hour, with fluorescence signals collected every minute. The results, shown in Figure 6, indicate that both 34°S-1 and 34°S-2 polymerases exhibit 5'-3' exo-cleavage activity.

[0080] Example 6

[0081] This example optimizes the PCR system.

[0082] Fourteen representative PCR reaction buffers were used to test the potential of 34°S-1 and 34°S-2 polymerases for PCR applications. The components of the 14 reaction buffers used are as follows:

[0083] Buffer 1: 10mM Tris-HCl (pH 8.4), 25mM KCl, 1.5mM MgCl2;

[0084] Buffer 2:10mM Tris-HCl(pH 8.4),25mM KCl,3.5mM MgCl2;

[0085] Buffer 3:10mM Tris-HCl(pH 8.4),75mM KCl,1.5mM MgCl2;

[0086] Buffer 4:10mM Tris-HCl(pH 8.4),75mM KCl,3.5mM MgCl2;

[0087] Buffer 5:10mM Tris-HCl(pH 8.8),25mM KCl,1.5mM MgCl2;

[0088] Buffer 6:10mM Tris-HCl(pH 8.8),25mM KCl,3.5mM MgCl2;

[0089] Buffer 7:10mM Tris-HCl(pH 8.8),75mM KCl,1.5mM MgCl2;

[0090] Buffer 8:10mM Tris-HCl(pH 8.8),75mM KCl,3.5mM MgCl2;

[0091] Buffer 9:10mM Tris-HCl(pH 9.2),25mM KCl,1.5mM MgCl2;

[0092] Buffer 10:10mM Tris-HCl(pH 9.2),25mM KCl,3.5mM MgCl2;

[0093] Buffer 11:10mM Tris-HCl(pH 9.2),75mM KCl,1.5mM MgCl2;

[0094] Buffer 12:10mM Tris-HCl(pH 9.2),75mM KCl,3.5mM MgCl2;

[0095] Buffer 13:20mM Tris-HCl(pH 8.8),10mM KCl,10mM KCl,10mM(NH4)2SO4,1mg / mL BSA,0.1% Triton;

[0096] Buffer 14: 20mM Tris-HCl (pH 8.8), 10mM KCl, 10mM KCl, 10mM (NH4)2SO4, 0.1% Triton.

[0097] 5× Additive: 200 mM TMAC, 2.5 M Betain, 12.5% ​​Glycerol, 0.25 mg / mL BSA, 0.5% Triton.

[0098] The Escherichia coli genome was used as a template for amplification of the 16S gene. The upstream and downstream primers used were E16S-27F and E16S-1492R, with primer sequences of 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO. 5) and 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO. 6), respectively. The PCR reaction system is shown in Table 4.

[0099] Table 4

[0100] The PCR cycle consisted of a 2-minute pre-denaturation at 95°C, followed by 30 cycles of 95°C for 20 seconds, 58°C for 30 seconds, and 72°C for 4 minutes, with a final reaction at 72°C for 5 minutes. The PCR products were subjected to agarose gel electrophoresis. Figure 7 shows the electrophoresis results of PCR amplification using 34°S-1 DNA polymerase in buffer 6, demonstrating that 34°S-1 DNA polymerase was capable of amplifying the target DNA in buffer 6. Figure 8 shows the electrophoresis results of PCR amplification using 34°S-2 DNA polymerase in different reaction buffers, demonstrating that 34°S-2 DNA polymerase was capable of amplifying the target DNA in various reaction buffers, with buffer 14 being the most suitable.

[0101] Example 7

[0102] In this example, inhibition resistance was measured.

[0103] Lambda DNA was used as a template for amplification of a 2 kb fragment. The upstream and downstream primers used were Anchor-λ F and λ-2 R (2 kb). The primer sequences were 5'-CCTGCTCTGCCGCTTCACGC-3' (SEQ ID NO. 7) and 5'-CCATGATTCAGTGTGCCCGTCTGG-3' (SEQ ID NO. 8), respectively. The PCR reaction system is shown in Table 5.

[0104] Table 5

[0105] The PCR cycle consisted of a 3-minute pre-denaturation at 95°C, followed by 30 cycles of 95°C for 30 seconds, 58°C for 30 seconds, and 72°C for 2 minutes, with a final reaction at 72°C for 5 minutes. The PCR products were subjected to agarose gel electrophoresis. The results are shown in Figure 9, demonstrating that 34°S-2 exhibits superior inhibition resistance to Taq. 34°S-2 can tolerate 20 μM hemin, 4 μg / mL humic acid, and 150 μg / mL tannic acid.

[0106] In summary, this application has discovered two new DNA polymerases, which have sequence similarities of only 40.92% and 41.94% with Taq DNA polymerase. They have a completely new sequence backbone and polymerase activity. In addition, they have higher thermal stability, 5'-3' exo-cleavage activity and inhibition resistance, providing a new polymerase backbone for the development of new molecular tools with a wider range of applications.

[0107] The applicant declares that while the above-mentioned embodiments are used to illustrate the detailed methods of the present application, the present application is not limited to the above-mentioned detailed methods, which does not mean that the present application must rely on the above-mentioned detailed methods in order to be implemented. Those skilled in the art should understand that any improvements to the present application, equivalent replacements for the raw materials of the present application's products, addition of auxiliary ingredients, and selection of specific methods, etc., fall within the scope of protection and disclosure of the present application.

Claims

1. A DNA polymerase, the amino acid sequence of which comprises: (1) the sequence shown in SEQ ID NO.1 or SEQ ID NO.2; or, (2) an amino acid sequence obtained by substituting, deleting or adding one or at least two amino acid residues to the sequence as described in (1), and having the same or similar function as the sequence as described in (1); or, (3) an amino acid sequence having at least 90% sequence homology with the sequence as described in (1) or (2), and having the same or similar function as the sequence as described in (1).

2. A nucleic acid molecule encoding the DNA polymerase according to claim 1.

3. The nucleic acid molecule according to claim 2, wherein The nucleic acid sequence of the nucleic acid molecule comprises the sequence shown in SEQ ID NO.3 or SEQ ID NO.

4.

4. A recombinant vector containing the nucleic acid molecule according to claim 2 or 3.

5. A recombinant cell containing the recombinant vector according to claim 4.

6. Use of the DNA polymerase according to claim 1 in the preparation of a nucleic acid amplification product.

7. Use of the nucleic acid molecule according to claim 2 or 3, the recombinant vector according to claim 4 or the recombinant cell according to claim 5 in the preparation of a DNA polymerase.

8. A nucleic acid amplification kit comprising the DNA polymerase according to claim 1.

9. Use of the DNA polymerase according to claim 1 in nucleic acid amplification.

10. A nucleic acid amplification method, comprising: Mix a nucleic acid template with a reaction system containing the DNA polymerase according to claim 1 and carry out an amplification reaction.

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