WO2025025231A1 - Dna polymerase and use thereof - Google Patents

Abstract

A DNA polymerase and the use thereof. The DNA polymerase is: 1) a protein having an amino acid sequence as shown in SEQ ID NO: 1 or 2; or 2) a protein having an amino acid sequence that has 80% or more, more preferably 90% or more, and further preferably 95% or more homology to the amino acid sequence as shown in SEQ ID NO: 1 or 2, and having a DNA polymerase activity. The DNA polymerase has a relatively high amplification rate, a relatively good thermal stability, and 5'-3' polymerization activity and 3'-5' exonuclease activity. During DNA amplification, the DNA polymerase can be used to perform DNA amplification at a relatively high PCR extension rate, thus being able to better meet market demand, and having a great application potential.

Description

DNA polymerase and its application Technical Field

The present invention relates to the field of biotechnology, and in particular to a DNA polymerase and an application thereof.

Background Art

DNA polymerase is an enzyme that uses a single strand of DNA as a template and four deoxynucleotides as substrates, starting from the 5’ end to replicate and synthesize a new DNA chain that is complementary to the sequence of the template chain. DNA polymerase can add free nucleotides to the 3’ end of the newly formed chain, resulting in the extension of the new chain in the 5’-3’ direction. Some enzymes have 3’ to 5’ exonuclease activity, which can correct errors in newly synthesized DNA. If mismatched bases are generated during PCR amplification, they can cut them off. After the wrong base is removed, the polymerase can reinsert the correct base and continue to replicate, thereby ensuring the accuracy of the amplification. In general, B family DNA polymerases all have correction activity. Compared with ordinary DNA polymerases (such as Taq DNA polymerase), they have a lower error rate and are more suitable for experiments with high PCR fidelity requirements, such as gene screening, sequencing, mutation detection, etc.

DNA polymerases are divided into six families: A, B, C, D, X, and Y. Most of the thermostable DNA polymerases discovered so far belong to the A family or the B family. The A family DNA polymerases are all derived from true bacteria, such as the Thermus or Bacillus. The B family thermostable DNA polymerases are all derived from archaea, such as the Thermococcus or Pyrococcus. The A family DNA polymerases mainly have 5’-3’ polymerization activity and 5’-3’ exonuclease activity. The main characteristics of the B family DNA polymerases are 5’-3’ polymerization activity and 3’-5’ nuclease exonuclease activity, and this unique exonuclease activity gives the B family DNA polymerases a proofreading function.

With the increasing application requirements, in addition to high amplification yield, more and higher requirements are put forward for the performance of DNA polymerases, such as high processivity, high amplification specificity, amplification performance for low-amount templates, high elongation rate, thermal stability, resistance to salt, and high fidelity. Among them, the A family DNA polymerase is represented by Taq DNA polymerase, which has high amplification efficiency but lacks fidelity; while among the B family DNA polymerases, most of the high-fidelity DNA polymerases are developed based on Pfu DNA polymerase or KOD DNA polymerase, which have disadvantages in having both high processivity and high fidelity performance. Therefore, finding new DNA polymerases is of great significance and value.

Summary of the invention

The main purpose of the present invention is to provide a DNA polymerase and its application, so as to solve the problem that the PCR amplification efficiency of the DNA polymerase in the prior art is difficult to exceed 1 kb/min.

In order to achieve the above-mentioned object, according to the first aspect of the present invention, a DNA polymerase is provided, which is: 1) a protein having an amino acid sequence as shown in SEQ ID NO: 1 or 2; or 2) a protein having an amino acid sequence with more than 80%, more preferably more than 90%, and further preferably more than 95% homology with the amino acid sequence as shown in SEQ ID NO: 1 or 2, and having DNA polymerase activity.

Furthermore, the T _m value of the DNA polymerase is 95.88° C.-96.45° C.; preferably, the amplification rate of the DNA polymerase is 1-4 kb/min; preferably, the amplification rate of the DNA polymerase is 3-4 kb/min; preferably, the DNA polymerase has 5'-3' polymerization activity and 3'-5' exolytic activity.

According to a second aspect of the present invention, a DNA molecule is provided, which encodes the above-mentioned DNA polymerase.

Furthermore, the DNA molecule is selected from: A) a polynucleotide consisting of a nucleotide sequence as shown in SEQ ID NO: 3 or 4; B) a polynucleotide having more than 80%, more preferably more than 90%, and further preferably more than 95% homology with a polynucleotide consisting of a nucleotide sequence as shown in SEQ ID NO: 3 or 4.

According to a third aspect of the present invention, a recombinant plasmid is provided, wherein the recombinant plasmid is connected to the above-mentioned DNA molecule.

According to a fourth aspect of the present invention, a host cell is provided, into which the above-mentioned recombinant plasmid is transfected.

Furthermore, the host cell includes a prokaryotic cell or a eukaryotic cell.

According to a fifth aspect of the present invention, there is provided a PCR amplification kit, comprising a DNA polymerase, wherein the DNA polymerase is the above-mentioned DNA polymerase.

Further, the PCR amplification kit further comprises: a PCR buffer, preferably the PCR buffer comprises a 10×PCR buffer, further preferably the 10×PCR buffer is selected from any one of the following: A) 150-200 mM Tris-HCl, 250-750 mM KCl, 1-15 mM MgCl ₂ , 1-4 mg/mL BSA and 100-600 mM TMAC at pH 8.2-8.6; B) 150-200 mM Tris-HCl, 250-750 mM KCl, 1-15 mM MgCl ₂ , 1-4 mg/mL BSA, 100-600 mM ammonium sulfate and 1-10% by volume of Tween 20 or NP40.

Furthermore, the PCR amplification kit further comprises: a PCR enhancer and dNTPs; preferably, the PCR enhancer comprises betaine.

According to a sixth aspect of the present invention, there is provided a method for performing PCR amplification using the above-mentioned DNA polymerase or the above-mentioned PCR amplification kit.

Furthermore, the PCR amplification rate is 1-4 kb/min; preferably, the PCR amplification rate is 3-4 kb/min.

According to a seventh aspect of the present invention, a method for constructing a sequencing library is provided, the method comprising: preparing a nucleic acid sample into a DNA fragment with a sequencing adapter; performing PCR amplification on the DNA fragment with the sequencing adapter to obtain a sequencing library; wherein the PCR amplification is performed using the above-mentioned DNA polymerase or the above-mentioned PCR amplification kit.

According to an eighth aspect of the present invention, a sequencing method is provided, the method comprising: performing sequencing on a sequencing library obtained by the above method to obtain a sequencing result.

By applying the technical solution of the present invention, the DNA polymerase of the present application has a higher amplification rate, better thermal stability, and 5'-3' polymerization activity and 3'-5' exo-cutting activity. When performing DNA amplification, DNA amplification can be performed at a faster PCR extension rate, which can better meet higher market demands and has greater application potential.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings constituting a part of the present application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

Figures 1A and 1B show the sequence comparison results of 27°N DNA polymerase and 4°S DNA polymerase with KOD DNA polymerase and Pfu DNA polymerase in Example 1 of the present invention;

Figure 2 shows the purification result of 27°N DNA polymerase in Example 2 of the present invention;

Figure 3 shows the purification result of 4°S DNA polymerase in Example 2 of the present invention;

FIG4 is a schematic diagram showing the principle of detecting the polymerization activity of a DNA polymerase in Example 4 of the present invention;

Figure 5 shows the results of the exo-activity assay of 27°N DNA polymerase, 4°S DNA polymerase and KOD DNA polymerase in Example 5 of the present invention;

Figure 6 shows the DNA electrophoresis diagram of 27°N DNA polymerase, 4°S DNA polymerase and Pfu DNA polymerase at different PCR amplification times in Example 6 of the present invention;

Figure 7 shows the DNA electrophoresis diagram of PCR amplification performed by 27°N DNA polymerase and 4°S DNA polymerase at different template contents in Example 7 of the present invention;

Figure 8 shows the DNA electrophoresis diagram of PCR amplification performed by 27°N DNA polymerase, 4°S DNA polymerase and Pfu DNA polymerase under different template lengths in Example 8 of the present invention.

DETAILED DESCRIPTION

It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

As mentioned in the background technology, among the DNA polymerases in the prior art, the A family DNA polymerases have high amplification efficiency, but do not have 3'-5' exonucleolytic activity, and the PCR fidelity is low. The B family DNA polymerases all have proofreading activity, which can ensure high fidelity. However, due to the increase in application requirements, the amplification efficiency and other properties of the DNA polymerases in the prior art, such as the ability to continuously synthesize, the amplification performance of low-amount templates, and thermal stability, cannot meet the current market demand. Therefore, this application intends to protect a DNA polymerase with both high continuous synthesis ability and high amplification efficiency.

In a first typical embodiment of the present application, a DNA polymerase is provided, which is: 1) a protein having an amino acid sequence as shown in SEQ ID NO: 1 or 2; or 2) a protein having an amino acid sequence with more than 80%, more preferably more than 90%, and further preferably more than 95% homology to the amino acid sequence as shown in SEQ ID NO: 1 or 2, and having DNA polymerase activity. The use of the above-mentioned DNA polymerase of the present application can complete DNA amplification with a high amplification efficiency on the basis of maintaining fidelity, and the sustained stability of the high amplification efficiency can meet the current application requirements for DNA polymerases.

SEQ ID NO: 1: (27°N DNA polymerase)

SEQ ID NO: 2: (4°S DNA polymerase)

As used herein, amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine (Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y) and valine (Val; V).

Substitution and replacement rules. Generally speaking, amino acids with similar properties will have similar effects after replacement. For example, conservative amino acid replacements may occur in the above homologous proteins. "Conservative amino acid replacements" include but are not limited to:

Hydrophobic amino acids (Ala, Cys, Gly, Pro, Met, Val, Ile, Leu) are replaced by other hydrophobic amino acids;

The hydrophobic amino acids with bulky side chains (Phe, Tyr, Trp) are replaced by other hydrophobic amino acids with bulky side chains;

Amino acids with positively charged side chains (Arg, His, Lys) are replaced by other amino acids with positively charged side chains;

Amino acids with polar, uncharged side chains (Ser, Thr, Asn, Gln) are replaced by other amino acids with polar, uncharged side chains.

A person skilled in the art may also perform conservative substitutions on amino acids according to amino acid substitution rules well known to those skilled in the art, such as the "blosum62 scoring matrix" in the prior art.

The above DNA polymerase has good application performance, such as amplification rate, amplification sensitivity, amplification extension and thermal stability. In a preferred embodiment, the Tm value of the DNA polymerase is 95.88°C-96.45°C, and it has high thermal stability; preferably, the amplification rate of the DNA polymerase is 1-4kb/min; preferably, the amplification rate of the DNA polymerase is 3-4kb/min, and it has high amplification efficiency; preferably, the DNA polymerase has 5'-3' polymerization activity and 3'-5' exo-cutting activity, which can ensure the fidelity of its PCR amplification process.

In a second typical embodiment of the present application, a DNA molecule is provided, which encodes the above-mentioned DNA polymerase.

In a preferred embodiment, the above-mentioned DNA molecule is selected from: A) a polynucleotide composed of a nucleotide sequence as shown in SEQ ID NO: 3 or 4; B) a polynucleotide having more than 80%, more preferably more than 90%, and further preferably more than 95% homology with a polynucleotide composed of a nucleotide sequence as shown in SEQ ID NO: 3 or 4.

SEQ ID NO: 3: (27°N DNA polymerase) (5'-3')

SEQ ID NO: 4: (4°S DNA polymerase) (5'-3')

Due to the principle of codon degeneracy, the nucleotide sequence for translating the amino acid sequence is not limited to the nucleotide sequence shown in SEQ ID NO: 3 or 4 above. Any nucleotide sequence that can encode the above amino acid sequence falls within the protection scope of the DNA molecule of this application.

In a third typical embodiment of the present application, a recombinant plasmid is provided, wherein the recombinant plasmid is connected to the above-mentioned DNA molecule.

The above DNA can encode the above DNA polymerase and can be connected to the recombinant plasmid to form a circular DNA. The above DNA and the recombinant plasmid can be transcribed and translated under the action of RNA polymerase, ribosome, tRNA, etc. to obtain the above DNA polymerase with high amplification efficiency and certain fidelity.

In a fourth typical embodiment of the present application, a host cell is provided, in which the above-mentioned recombinant plasmid is transfected. In a preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell. Specifically, the prokaryotic cell can be Escherichia coli, and the eukaryotic cell can be yeast.

By using the above host cells, the recombinant plasmid can be replicated in the host cells, and the DNA molecules carried on the recombinant plasmid can also be transcribed and translated to obtain a large amount of DNA polymerase. By using the existing technology, the host cells can be crushed for protein purification, crushed and then catalyzed by crude enzymes or other methods to obtain DNA polymerase and perform subsequent DNA amplification. The host cell is a host cell of non-plant origin.

In a fifth typical embodiment of the present application, a PCR amplification kit is provided, comprising a DNA polymerase, wherein the DNA polymerase is the above-mentioned DNA polymerase. The kit can be used to conveniently and quickly perform a DNA amplification reaction with high efficiency and good fidelity, and has good application prospects.

To further ensure the amplification efficiency of the DNA polymerase participating in the amplification reaction, in a preferred embodiment, the PCR amplification kit further includes: a PCR buffer, preferably a PCR buffer including a 10×PCR buffer, any PCR buffer concentration suitable for the DNA amplification reaction is applicable to the present application, in a preferred embodiment, the PCR buffer includes a 1×PCR buffer, a 10×PCR buffer or a 20×PCR buffer. It is further preferred that the 10×PCR buffer is selected from any one of the following: A) pH 8.2-8.6 150-200mM Tris-HCl, 250-750mM KCl, 1-15mM MgCl ₂ , 1-4mg/mL BSA and 100-600mM TMAC. Among them, TMAC can increase the efficiency of the PCR reaction and improve the specificity of the PCR reaction. B) pH 8.2-8.6 150-200 mM Tris-HCl, 250-750 mM KCl, 1-15 mM MgCl ₂ , 1-4 mg/mL BSA, 100-600 mM ammonium sulfate and 1-10% Tween 20 or NP40. Among them, ammonium sulfate can release the non-specific binding between primers and templates and improve the specificity of the reaction, while Tween 20 or NP40 has a certain protective effect on the enzymes in the PCR reaction.

In order to further ensure the amplification efficiency, amplification sensitivity and fidelity of the DNA polymerase participating in the amplification reaction, in a preferred embodiment, the PCR amplification kit also includes: a PCR enhancer and dNTPs. Among them, the PCR enhancer can improve the sensitivity, specificity and fidelity of the DNA amplification process, and improve amplification errors such as false positives and false negatives. Any type of PCR enhancer is suitable for this application, including but not limited to small molecule compounds, proteins or nanomaterials. In a preferred embodiment, the PCR enhancer includes betaine.

In a sixth typical embodiment of the present application, a method for PCR amplification using the above-mentioned DNA polymerase or the above-mentioned PCR amplification kit is provided.

The DNA polymerase of the present application has a high amplification efficiency. In a preferred embodiment, the PCR amplification rate is 1-4 kb/min; preferably, the PCR amplification rate is 3-4 kb/min.

In a seventh typical embodiment of the present application, a method for constructing a sequencing library is provided, the method comprising: preparing a nucleic acid sample into a DNA fragment with a sequencing adapter; performing PCR amplification on the DNA fragment with the sequencing adapter to obtain a sequencing library; wherein, performing PCR amplification using the above-mentioned DNA polymerase or the above-mentioned PCR amplification kit can obtain a sequencing library with better quality in a shorter time.

The sample types for sequencing library construction by the DNA polymerase of the present application include DNA and RNA. If the sample is RNA, it is necessary to reverse transcribe the sample to obtain cDNA for subsequent library construction before sequencing adapters are made. In addition, it is possible to choose whether to fragment the DNA according to the fragment size of the sample, including physical shearing and chemical shearing methods; and to choose whether to perform end repair on the DNA fragments before connecting the DNA sequencing adapters according to the type of adapters used to construct the library.

In an eighth typical embodiment of the present application, a sequencing method is provided, the method comprising: performing sequencing on a sequencing library obtained by the above method to obtain a sequencing result.

The application of the DNA polymerase of the present application is not limited to high-throughput sequencing methods. So-called "high-throughput sequencing methods" include "second generation" and/or "third generation" sequencing methods. These high-throughput sequencing methods can provide single-molecule sequencing, and adopt technologies such as pyrophosphate sequencing, reversible terminator sequencing, sequencing by cleavable probes connected, sequencing by non-cleavable probes connected, DNA nanoballs, and real-time single-molecule sequencing.

The present application is further described in detail below in conjunction with specific embodiments. These embodiments should not be construed as limiting the scope of protection claimed in the present application.

Example 1 Sequence Alignment

The Clustal Omega online sequence alignment website was used to perform multiple sequence alignment of the two novel DNA polymerases disclosed in the present invention (27°N DNA polymerase and 4°S DNA polymerase), KOD DNA polymerase (as shown in SEQ ID NO: 14), and Pfu DNA polymerase (as shown in SEQ ID NO: 15). The alignment results showed that the sequence identities of 27°N DNA polymerase, KOD, and Pfu DNA polymerase were 43.41% and 44.95%, respectively. The sequence identities of 4°S DNA polymerase, KOD, and Pfu DNA polymerase were 42.61% and 43.62%, respectively. The specific alignment results are shown in Figures 1A and 1B.

SEQ ID NO: 14: KOD DNA polymerase

SEQ ID NO: 15: Pfu DNA polymerase

Example 2 Construction of recombinant plasmid, expression and purification

Changzhou Xinyisheng Life Science Co., Ltd. was commissioned to synthesize the gene sequences of 27°N DNA polymerase and 4°S DNA polymerase, and the genes were cloned into the pET28a expression vector with cloning sites of Nde I and Xho I. The above recombinant plasmids were transformed into Escherichia coli BL21 (DE3) competent cells and kept at 37°C overnight for subsequent expression purification.

1. The affinity chromatography column and cation exchange column used for 27°N DNA polymerase protein purification are HisTrap FF 5mL and HiTrap SP HP 5mL respectively. The specific purification steps are as follows:

1. Pick 3-6 single colonies with good growth on the plate, inoculate them into a 50/250ml LB liquid conical flask, and culture at 37℃ for 5-7h until _OD600 reaches 0.6-4.0. Then inoculate the above bacterial liquid into 2L/5L LB medium at a 1% inoculation amount, culture at 37℃ for 2-4h, and wait until _OD600 reaches 0.8-1.0. Precool the original shaker to 16℃. Add IPTG to the culture medium to a final concentration of 0.5mM. Induce expression for 12-16h in a shaker at 16℃, 220rpm.

2. Collect the cells by centrifugation at 8000g for 30 minutes, then add affinity solution A at a ratio of 1:10 to resuspend the cells, and use ultrasound to break the cells in an ice bath environment.

3. Heating treatment: Preheat the water bath to 75-80℃, put the broken bacteria into the water bath, shake and mix, use a clean thermometer to detect the internal temperature of the bacterial solution. When it reaches 75℃, start timing for 30 minutes. During this period, shake and mix 3 times every 10 minutes to ensure uniform heating.

4. Centrifuge the heat-treated ultrasonic disrupted liquid at 12000 rpm and 4°C for 60 min, and filter the supernatant with a 0.22 μM filter membrane as the sample for loading the purification column.

5. Load the above sample into the pretreated chromatography column (HisTrap FF) at a rate of 3 ml/min. After loading, continue to rinse with Ni column affinity A solution for 20 column volumes. Then perform a linear elution of 10.5 CV with Ni column affinity B solution accounting for 0-70%. Collect the eluted protein when the UV absorption peak reaches 50 mAu, and stop collecting when the UV absorption peak drops to 200 mAu.

6. Dilute the collected eluate 10 times with diluent and load it onto the pretreated SP column (HiTrap SP HP 5ml). After loading, rinse with SP column A solution for 30CV until the baseline is stable, with a flow rate of 5min/min.

7. SP column B solution was used for gradient elution (0-70% SP column B solution, 20CV) of the target protein at a flow rate of 5 ml/min. The collected samples were analyzed by SDS-PAGE to determine the protein purity.

8. After the purified protein sample is dialyzed and the concentration is determined, it is stored in storage solution for subsequent functional activity analysis.

The specific components of the buffer used in the purification process are shown below, and the purification results are shown in Figure 2:

Ni column-A Buffer: 20mM Tris-HCl (2.42g/L), 500mM NaCl (29.22g/L), 20mM Imid azole (1.36g/L), 5% Glycerol (62.5g/L), pH 7.5;

Ni column-B Buffer: 20mM Tris-HCl (2.42g/L), 500mM NaCl (29.22g/L), 500mM Imid azole (34.04g/L), 5% Glycerol (62.5g/L), pH 7.5;

SP column-A Buffer: 20mM Tris-Hcl (2.42g/L), 50mM NaCl (2.92g/L), 5% Glycerol (62.5g/L), pH 7.5;

SP column-B Buffer: 20mM Tris-Hcl (2.42g/L), 1M NaCl (58.44g/L), 5% Glycerol (62.5g/L), pH 7.5;

Diluent: 20 mM Tris-HCl (2.42 g/L), 5% Glycerol (62.5 g/L), pH 7.5;

2X dialysate: 40mM Tris-HCl, 200mM KCl, 2mM DTT (0.154g/L), 0.2mM EDTA (0.0884g/L), 5% Glycerol, pH 8.0@25℃;

Storage solution: 10mM Tris-HCl (1.2114g/L), 100mM KCl (7.455g/L), 1mM DTT (0.15425g/L), 0.1mM EDTA (0.037224g/L), 50% Glycerol (625g/L), pH 7.5@25℃.

2. The affinity chromatography column and anion exchange column used for purification of 4°S DNA polymerase protein are HisTrap FF 5mL and HiTrap Q HF 5mL respectively. The specific purification steps are as follows:

1. Pick 3-6 single colonies with good growth on the plate, inoculate them into a 50/250ml LB liquid conical flask, and culture at 37℃ for 5-7h until _OD600 reaches 0.6-4.0. Then inoculate the above bacterial liquid into 2L/5L LB medium at a 1% inoculation amount, culture at 37℃ for 2-4h, and wait until _OD600 reaches 0.8-1.0. Precool the original shaker to 16℃. Add IPTG to the culture medium to a final concentration of 0.5mM. Induce expression for 12-16h in a 16℃ shaker at 220rpm.

2. Collect the cells by centrifugation at 8000g for 30 minutes, then add affinity solution A at a ratio of 1:10 to resuspend the cells, and use ultrasound to break the cells in an ice bath environment.

3. Heating treatment: Preheat the water bath to 75-80℃, put the broken bacteria into the water bath, shake and mix, use a clean thermometer to detect the internal temperature of the bacterial solution. When it reaches 75℃, start timing for 30 minutes. During this period, shake and mix 3 times every 10 minutes to ensure uniform heating.

4. Centrifuge the heat-treated ultrasonic disrupted liquid at 12000 rpm and 4°C for 60 min, and filter the supernatant with a 0.22 μM filter membrane as the sample for loading the purification column.

5. Load the above sample into the pretreated chromatography column (HisTrap FF 5mL) at a rate of 3ml/min. After loading, continue to rinse with Ni column affinity A solution for 20 column volumes. Then perform linear elution with Ni column affinity B solution accounting for 0-70% and 10.5CV. Collect the eluted protein when the UV absorption peak reaches 50mAu, and stop collecting when the UV absorption peak drops to 200mAu.

6. Dilute the collected eluate 6 times with diluent and load it onto the pretreated Q column (HiTrap Q HP 5ml). After loading, rinse the Q column with solution A for 30CV until the baseline is stable, with a flow rate of 5min/min.

7. The target protein was eluted with Q column solution B (0-100% Q column solution B, 10CV) at a flow rate of 5 ml/min. The collected samples were analyzed by SDS-PAGE to determine the purity of the protein.

8. After the purified protein sample is dialyzed and the concentration is determined, it is stored in storage solution for subsequent functional activity analysis.

The specific components of the buffer used in the purification process are shown below, and the purification results are shown in Figure 3:

Ni column-A Buffer: 20mM Tris-HCl, 300mM NaCl, 20mM Imidazole, 5% Glycerol, pH 8.5;

Ni column-B Buffer: 20mM Tris-HCl, 300mM NaCl, 500mM Imidazole, 5% Glycerol, pH 8.5;

Q column-A Buffer: 20mM Tris-HCl, 100mM NaCl, 5% Glycerol, pH 8.5;

Q column-B Buffer: 20mM Tris-HCl, 500mM NaCl, 5% Glycerol, pH 8.5;

Diluent: 20 mM Tris-HCl, 5% Glycerol, pH 8.5;

2X dialysate: 40mM Tris-HCl, 200mM KCl, 2mM DTT (0.154g/L), 0.2mM EDTA (0.0884g/L), 5% Glycerol, pH 8.0@25℃;

Storage solution: 10mM Tris-HCl (1.2114g/L), 100mM KCl (7.455g/L), 1mM DTT (0.15425g/L), 0.1mM EDTA (0.037224g/L), 50% Glycerol (625g/L), pH 8.0@25℃.

Example 3 Thermal Stability Determination

Protein Thermal ShiftTM dye kit (ThermoFisher) was used to determine protein stability, and KOD DNA polymerase and Pfu DNA polymerase were used as controls. The test reaction system was: 5μL Protein Thermal Shift Buffer, 2μL test protein (purity greater than 90%, concentration 0.2mg/ml-2mg/ml), 2.5μL 8×Protein thermal shift dye, 10.5μL NF water. The above reaction system was mixed and placed on a qPCR instrument for a 25-99°C temperature increase experiment, and the changes in ROX fluorescence signals were monitored. The results showed that the two novel DNA polymerases disclosed in the present invention had good thermal stability, and their T _m values were comparable to those of KOD DNA polymerase and higher than those of Pfu DNA polymerase, as shown in Table 1 below:

Table 1:

Example 4 Polymerization Activity Determination

Primed M13 ssDNA substrate was used for polymerization activity determination. The specific principle is shown in Figure 4. In the presence of polymerization activity, the primer on the primed M13 ssDNA will extend along the ssDNA in the 5’-3’ direction to produce dsDNA, which can be quantitatively detected using Qubit dsDNA HS Assay Kits.

The specific reaction system and components are shown in Table 2 below:

Table 2:

After the above reaction solution was reacted at 72℃ for 5 minutes, 2μl 0.5M EDTA was added to terminate the reaction, and the dsDNA concentration was determined using the Qubit kit. The negative control NC is a reaction system without polymerase. The results are shown in Table 3 below (the Qubit value is the value after subtracting the NC control):

Table 3:

The above results show that the two novel B-family DNA polymerases disclosed in the present invention have polymerization activity at 72°C.

Example 5 Exo-activity assay (3'-5' exo-activity)

The end mismatch fluorescent probe method was used to qualitatively confirm the exolytic activity of 27°N DNA polymerase and 4°S DNA polymerase (reaction at 37°C for 1h). The probe sequences were ATCAGCAGGCCACACGTTAAACTGT-BHQ2 (SEQ ID NO: 5) and FAM-5’-TGTCTTTAACGTGTGGCCTGCTGAT (SEQ ID NO: 6).

The reaction system used for the exosome activity assay is as follows: 2.5 μL Reaction Buffer, 0.25 μL of 10 μM fluorescent probe substrate, 2 μL of enzyme solution (the enzyme concentration used was 0.6 mg/ml), and 20.25 μL of NF water.

The negative control NC group also used the above reaction system, and the enzyme solution components were replaced with the corresponding enzyme storage solution. The positive control PC group also used the above reaction system, in which the enzyme was the currently known B family DNA polymerase KOD. The above reaction system was configured on ice, the entire reaction system was 25 μL, the reaction was carried out in a 384-well plate, and then placed in a microplate reader for fluorescence signal detection, with the excitation light and emission light set to 492 nm and 518 nm, respectively. The fluorescence signal was collected every 30 seconds, and the entire reaction time was 1 hour.

The results are shown in Figure 5 (the corresponding blank control values have been subtracted from the fluorescence values): The line segments in Figure 5 are the changes in the fluorescence signals monitored in real time during the experiment, so the line segments are connected in a zigzag manner and do not appear as straight lines. The results show that similar to KOD DNA polymerase, both 27°N DNA polymerase and 4°S DNA polymerase have 3'-5' exonuclease activity, which can ensure the correction function of DNA polymerase during the PCR process.

Example 6 PCR performance test (PCR amplification extension rate test)

In order to test the amplification rate of 27°N DNA polymerase and 4°S DNA polymerase in PCR applications, the following reaction system was used for PCR amplification experiments. Specifically, different extension times were used to amplify the 2kb target gene fragment, and the amplification effect was detected by agarose gel electrophoresis. The template substrate used was λDNA, and the amplification primers used were:

λF: CCTGCTCTGCCGCTTCACGC (SEQ ID NO: 7)

λ-2R(2kb)：CCATGATTCAGTGTGCCCGTCTGG(SEQ ID NO：8)

The PCR reaction system is shown in Table 4 below:

Table 4:

The 10× reaction buffer used for 27°N DNA polymerase amplification is: 200 mM Tris-HCl (pH 8.4), 250 mM KCl, 15 mM MgCl ₂ , 3.6 mg/ml BSA, 100 mM ammonium sulfate, and 2% Tween 20/NP40.

The 10× reaction buffer used for 4°S DNA polymerase amplification was: 150 mM Tris-HCl (pH 8.4), 750 mM KCl, 1 mM MgCl ₂ , 3.6 mg/ml BSA, and 100 mM TMAC.

The PCR reaction system is shown in Table 5 below:

Table 5:

The PCR amplification results are shown in Figure 6. It can be seen that 27°N DNA polymerase can amplify a relatively clear main target band when the extension time is 30s. 27°N DNA polymerase has a higher PCR amplification rate, and can amplify a relatively clear target band in 30s. The amplification rate is about 3 times that of pfu DNA polymerase. 4°S DNA polymerase takes about 90s to amplify a 2kb target band, and the amplification rate is about greater than 1kb/min. Both new DNA polymerases disclosed in the present invention have good PCR amplification rates.

Example 7 PCR performance test (sensitivity of PCR amplification)

In order to test the amplification sensitivity of 27°N DNA polymerase and 4°S DNA polymerase in PCR reaction, PCR amplification was performed using the reaction system in Table 6 below under the conditions of different substrate addition amounts:

Table 6:

The forward primer λF used in the above reaction system is CCTGCTCTGCCGCTTCACGC (SEQ ID NO: 7), and λ-2R (2kb) is CCATGATTCAGTGTGCCCGTCTGG (SEQ ID NO: 8).

The PCR amplification program is shown in Table 7 below:

Table 7:

The PCR amplification results are shown in Figure 7. When the substrate template concentration is 100pg or above, 27°N DNA polymerase can specifically amplify the target band. While 4°S DNA polymerase can specifically amplify the corresponding target band under the condition of 10pg substrate template concentration. Both of the two novel DNA polymerases disclosed in the present invention have good amplification sensitivity and have a large room for improvement.

Example 8 PCR performance test (amplification of target gene fragments of different lengths)

In order to test the amplification ability of 27°N DNA polymerase and 4°S DNA polymerase, the following reaction system was used for PCR amplification experiment to amplify target gene fragments of different lengths. The template substrate used was λDNA, and the amplification primers used were:

λF: CCTGCTCTGCCGCTTCACGC (SEQ ID NO: 7)

λ-2R (2kb): CCATGATTCAGTGTGCCCGTCTGG (SEQ ID NO: 8)

λ-4R(4kb)：CCAGGACTATCCGTATGACTACG(SEQ ID NO：9)

λ-6R(6kb)：GAGATGGCATATTGCTACGCAAGA(SEQ ID NO：10)

λ-8R(8kb)：GCCTCGTTGCGTTTTGTTTGCACG(SEQ ID NO：11)

λ-10R(10kb)：GCACAGAAGCTATTATGCGTCCCCAGG(SEQ ID NO：12)

λ-12R(12kb)：TCTTCCTCGTGCATCGAGCTATTCGG(SEQ ID NO：13)

Using λDNA as template, the above primers were used to amplify target gene fragments of 2kb, 4kb, 6kb, 8kb, 10kb, and 12kb, respectively. The PCR reaction system is shown in Table 8 below:

Table 8:

The 10× reaction buffer used for 27°N DNA polymerase amplification is: 200 mM Tris-HCl (pH 8.4), 250 mM KCl, 15 mM MgCl ₂ , 3.6 mg/mL BSA, 100 mM ammonium sulfate, and 2% Tween 20/NP40.

The 10× reaction buffer used for 4°S DNA polymerase amplification was: 150 mM Tris-HCl (pH 8.4), 750 mM KCl, 1 mM MgCl ₂ , 3.6 mg/mL BSA, and 100 mM TMAC.

The PCR reaction system is shown in Table 9 below:

Table 9:

The PCR amplification results are shown in Figure 8. Both 27°N DNA polymerase and 4°S DNA polymerase can amplify the corresponding target bands. However, when amplifying target gene fragments larger than 8 kb, the yield of the target amplification product is low.

From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: the DNA polymerase of the present application has a high amplification rate, good thermal stability and amplification sensitivity, can amplify target gene fragments of different lengths, and has 5'-3' polymerization activity and 3'-5' exonuclease activity. When performing DNA amplification, DNA amplification can be performed at a faster PCR extension rate, which can better meet higher market demands and has great application potential.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims (14)

A DNA polymerase, characterized in that the DNA polymerase is: 1) a protein having an amino acid sequence as shown in SEQ ID NO: 1 or 2; or 2) A protein having an amino acid sequence that is more than 80%, more preferably more than 90%, and even more preferably more than 95% homologous to the amino acid sequence shown in SEQ ID NO: 1 or 2, and having DNA polymerase activity. The DNA polymerase according to claim 1, characterized in that the T _m value of the DNA polymerase is 95.88°C-96.45°C; Preferably, the amplification rate of the DNA polymerase is 1-4 kb/min; Preferably, the amplification rate of the DNA polymerase is 3-4 kb/min; Preferably, the DNA polymerase has 5’-3’ polymerization activity and 3’-5’ exonuclease activity. A DNA molecule, characterized in that the DNA molecule encodes the DNA polymerase according to claim 1 or 2. The DNA molecule according to claim 3, characterized in that the DNA molecule is selected from: A) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 3 or 4; B) A polynucleotide having more than 80%, more preferably more than 90%, and further preferably more than 95% homology with the polynucleotide consisting of the nucleotide sequence as shown in SEQ ID NO: 3 or 4. A recombinant plasmid, characterized in that the recombinant plasmid is connected to the DNA molecule according to claim 3 or 4. A host cell, characterized in that the recombinant plasmid according to claim 5 is transfected into the host cell. The host cell according to claim 6, characterized in that the host cell comprises a prokaryotic cell or a eukaryotic cell. A PCR amplification kit, comprising a DNA polymerase, characterized in that the DNA polymerase is the DNA polymerase according to claim 1 or 2. The PCR amplification kit according to claim 8, characterized in that the PCR amplification kit further comprises: a PCR buffer, preferably the PCR buffer comprises a 10×PCR buffer, and further preferably the 10×PCR buffer is selected from any one of the following: A) pH 8.2-8.6 150-200 mM Tris-HCl, 250-750 mM KCl, 1-15 mM MgCl ₂ , 1-4 mg/mL BSA and 100-600 mM TMAC; B) pH 8.2-8.6: 150-200 mM Tris-HCl, 250-750 mM KCl, 1-15 mM MgCl ₂ , 1-4 mg/mL BSA, 100-600 mM ammonium sulfate, and 1-10% Tween 20 or NP40. The PCR amplification kit according to claim 8, characterized in that the PCR amplification kit further comprises: a PCR enhancer and dNTPs; Preferably, the PCR enhancer comprises betaine. A method for PCR amplification using the DNA polymerase according to claim 1 or 2 or the PCR amplification kit according to any one of claims 8 to 10. The method according to claim 11, characterized in that the PCR amplification rate is 1-4 kb/min; Preferably, the PCR amplification rate is 3-4 kb/min. A method for constructing a sequencing library, characterized in that the method comprises: preparing nucleic acid samples into DNA fragments with sequencing adapters; Amplifying the DNA fragment with the sequencing adapter by PCR to obtain the sequencing library; Wherein, the PCR amplification is performed using the DNA polymerase described in claim 1 or 2 or the PCR amplification kit described in any one of claims 8 to 10. A sequencing method, characterized in that the method comprises: The sequencing library obtained by the method according to claim 13 is sequenced on a sequencing machine to obtain a sequencing result.