Phi29 DNA polymerase mutant having reduced polymerization rate, preparation method therefor, and use thereof
By modifying the amino acid sequence of phi29 DNA polymerase, a mutant phi29 DNA polymerase with a reduced polymerization rate was prepared, which solved the sequencing accuracy and quality problems caused by excessively fast polymerization rate and improved the accuracy and stability of DNA amplification and sequencing.
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- CIXI INST OF BIOMEDICAL ENG NINGBO INST OF IND TECH CHINESE ACAD OF SCI NINGBO
- Filing Date
- 2025-01-09
- Publication Date
- 2026-07-02
Smart Images
Figure CN2025071408_02072026_PF_FP_ABST
Abstract
Description
A mutant of phi29 DNA polymerase with reduced polymerization rate, its preparation method and application Technical Field
[0001] This invention belongs to the field of biotechnology and relates to a phi29 DNA polymerase mutant with reduced polymerization rate, its preparation method, and its application. Background Technology
[0002] Phi29 DNA polymerase, derived from bacteriophage phi 29 of Bacillus subtilis, is an important enzyme with outstanding properties. It not only possesses highly efficient and continuous synthesis capabilities and strong strand substitution ability, but can also synthesize DNA fragments up to 70kb in length. This makes it an ideal choice for in vitro isothermal DNA amplification processes, eliminating the need for traditional thermal cycling methods. The applications of Phi29 DNA polymerase are extremely wide-ranging, including but not limited to rolling circle amplification (RCA), multiple substitution amplification (MDA), amplification of pathogen genomes and metagenomics, detection of viruses and miRNAs, and sequencing analysis.
[0003] In the context of the development of third-generation sequencing technology, the importance of Phi29 DNA polymerase and its analogues is particularly prominent. Third-generation sequencing technology, with its significant characteristics such as high throughput and long reads, is changing the way we study genomes. The core of this technology lies in the principle of "sequencing while synthesizing," which achieves sequence determination by real-time monitoring of the addition of bases during the synthesis of new DNA strands by the DNA polymerase. In this process, the performance of the polymerase directly affects the success of sequencing. To ensure high-quality sequencing results, in addition to requiring the polymerase to have strong strand substitution capabilities and continuous synthesis capacity, strict requirements are also placed on its polymerization rate.
[0004] Excessively high polymerase rates can have several drawbacks. First, the sequencer may not be able to accurately record each base addition in a timely manner, affecting sequencing accuracy. Second, in cases where the DNA template has a complex structure or is damaged, a rapid synthesis rate can increase the probability of mismatches between the polymerase and the template, thus reducing the quality of sequencing data and blurring the line between false positives and results caused by polymerase errors. Furthermore, a major advantage of third-generation sequencing technology is its ability to directly detect chemical modifications on DNA, such as methylation. This process relies on the subtle pauses that occur when the polymerase encounters modified bases. However, if the polymerization rate is too high, these important pause signals may not be effectively captured, thus diminishing the technology's application value in epigenetic research.
[0005] Therefore, to maximize the advantages of third-generation sequencing technology and ensure the accuracy and reliability of sequencing results, it is essential to carefully regulate the polymerization rate of DNA polymerases involved in the sequencing reaction, finding an optimal balance between sequencing efficiency and high-quality output. This is of great significance for promoting the development of sequencing technology and deepening our understanding of life sciences. Technical issues
[0006] The purpose of this invention is to address the aforementioned problems in the prior art by proposing a phi29 DNA polymerase mutant with reduced polymerization rate, which reduces the polymerization rate of the polymerase while maintaining good polymerase activity. Technical solutions
[0007] The objective of this invention can be achieved through the following technical solution: a phi29 DNA polymerase mutant with reduced polymerization rate, wherein the phi29 DNA polymerase mutant includes any one of A1) to A2):
[0008] A1) An amino acid sequence as shown in any one of SEQ ID NO. 54 to SEQ ID NO. 104;
[0009] A2) An amino acid sequence obtained by attaching a tag to the middle and / or N-terminus and / or C-terminus of the amino acid sequence shown in A1).
[0010] The tags described in this invention include at least one type of tag that facilitates the dissolution, expression, purification, fixation, and detection of phi29 DNA polymerase mutants.
[0011] The tag includes at least one of the tags that facilitate the dissolution, purification, and detection of the phi29 DNA polymerase mutant. It is understood that the phi29 DNA polymerase mutant of the present invention may contain one or more tags; multiple tags may comprise a combination of multiple identical tags, or a combination of multiple different tags.
[0012] For example, tags that facilitate the dissolution of phi29 DNA polymerase mutants include, but are not limited to, nus tags or maltose-binding proteins; tags that facilitate the purification of phi29 DNA polymerase mutants include, but are not limited to, strep tags, His tags, GST tags, pelB signal sequences, or ompA signal sequences; tags that facilitate the detection of phi29 DNA polymerase mutants include, but are not limited to, horseradish peroxidase (HRP), β-galactosidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, or cyan fluorescent protein (CFP).
[0013] Preferably, the label is a strep label.
[0014] In the above-mentioned phi29 DNA polymerase mutant, the phi29 DNA polymerase mutant has an amino acid sequence that is at least 90% identical to the amino acid sequence shown in any one of SEQ ID NO. 54 to SEQ ID NO. 104.
[0015] Preferably, the phi29 DNA polymerase mutant is an amino acid residue sequence of a protein derived from Bacillus subtilis that has more than 90% identity with the amino acid sequence shown in any one of SEQ ID NO. 54 to SEQ ID NO. 104.
[0016] Further preferred, the phi29 DNA polymerase mutant is an amino acid residue sequence of a protein derived from Bacillus subtilis that has more than 95% identity with the amino acid sequence shown in any one of SEQ ID NO.54 to SEQ ID NO.104.
[0017] Biological materials related to the aforementioned phi29 DNA polymerase mutant are also within the scope of protection of this invention, and the biological materials are any one of B1) to B4):
[0018] B1) The nucleic acid molecule encoding the above-mentioned phi29 DNA polymerase mutant;
[0019] B2), an expression cassette containing the nucleic acid molecule described in B1);
[0020] B3) A recombinant vector containing the nucleic acid molecule described in B1) or the expression cassette described in B2);
[0021] B4) Recombinant biological cells containing the nucleic acid molecule described in B1), the expression cassette described in B2), or the recombinant vector described in B3).
[0022] The recombinant vector contains the nucleic acid molecule described in B1) or the expression cassette described in B2), and the recombinant biological cell contains the nucleic acid molecule described in B1), the expression cassette described in B2), or the recombinant vector described in B3).
[0023] In the above-mentioned biological materials, the nucleotide sequence of the nucleic acid molecule is shown in any one of SEQ ID NO.1 to SEQ ID NO.51.
[0024] Preferably, the expression cassette is DNA expressing the phi29 DNA polymerase mutant in host cells. This DNA may include not only a promoter to initiate transcription of the phi29 DNA polymerase mutant gene, but also a terminator to terminate transcription of the protein gene. Furthermore, the expression cassette may also include an enhancer sequence.
[0025] Preferably, the recombinant vector is a recombinant vector obtained by inserting a nucleic acid molecule encoding the phi29 DNA polymerase mutant into the multiple cloning site of a biological vector.
[0026] Preferably, the biological vector is at least one of plasmids, granules, bacteriophages, and viral vectors. Specifically, it can be the PET-28a vector.
[0027] Preferably, biological cells include prokaryotic cells and eukaryotic cells.
[0028] The prokaryotic cells include at least one of bacteria and algae.
[0029] The eukaryotic cells include at least one of fungi, mammalian cells, and insect cells. The bacteria may be *Escherichia coli*, such as *Escherichia coli* BL21(DE3).
[0030] The recombinant organism does not contain reproductive material.
[0031] The recombinant biological cell is a recombinant biological cell obtained by introducing the nucleic acid molecule described in B1), the expression cassette described in B2), or the recombinant vector described in B3) into a biological cell.
[0032] Specifically, it can be recombinant Escherichia coli obtained by introducing a recombinant vector into Escherichia coli BL21(DE3).
[0033] The present invention also provides the application of primer combinations in amplifying nucleic acid molecules encoding the above-mentioned phi29 DNA polymerase mutant.
[0034] The present invention also provides an enzyme preparation comprising the above-described phi29 DNA polymerase mutant.
[0035] The present invention also provides a method for preparing the above-mentioned phi29 DNA polymerase mutant, the method comprising the following steps: introducing the coding gene of the above-mentioned phi29 DNA polymerase mutant into a biological cell, so that the coding gene is expressed, thereby obtaining the phi29 DNA polymerase mutant.
[0036] The present invention also provides a method for purifying the above-mentioned phi29 DNA polymerase mutant, the method comprising the following steps: high-throughput purification of the above-mentioned phi29 DNA polymerase mutant using strep-tagged protein purification magnetic beads.
[0037] The present invention also provides a method for amplifying or sequencing template DNA, the method comprising the following steps: amplifying or sequencing template DNA using the above-mentioned phi29 DNA polymerase mutant.
[0038] In the above-mentioned method for amplifying or sequencing template DNA, a labeled nucleotide substrate is used during the amplification or sequencing process. The structural formula of the labeled nucleotide substrate is as follows:
[0039] .
[0040] The present invention also provides the application of any one of C1) to C3) in any one of D1) to D4), wherein C1) to C3) and D1) to D4) are as follows:
[0041] C1) The above-mentioned phi29 DNA polymerase mutant;
[0042] C2), the aforementioned biological materials;
[0043] C3), the enzyme preparations mentioned above;
[0044] D1) Nucleic acid amplification;
[0045] D2) Preparation of nucleic acid amplification-related products;
[0046] D3), sequencing;
[0047] D4) Prepare sequencing-related products.
[0048] The present invention also provides a nucleic acid amplification or sequencing related product, including the above-mentioned phi29 DNA polymerase mutant or the above-mentioned enzyme preparation.
[0049] In one of the aforementioned nucleic acid amplification or sequencing-related products, the product also includes non-natural substrates. Beneficial effects
[0050] Compared with the prior art, the present invention has the following beneficial effects: The present invention designs and provides a mutant of phi29 DNA polymerase. By modifying at least one amino acid residue in the amino acid sequence of phi29 DNA polymerase (190, 324, 411, 49, 83, 375, 526, 85, 149, 284, 287, 35, 115, 178, 102, 246, 336, 506, 257, 502, 55, 113, 187, 289, 306, 517, 527, 140, 203, 368, 372, 89, 54, 277, 327, 163, 224), a novel protein is generated that reduces the polymerization rate of the polymerase while maintaining good polymerase activity, successfully solving the problem of phi29. The problems caused by excessively fast polymerization rates in practical applications of DNA polymerase have been addressed by improving its accuracy, stability, and reliability in DNA amplification, sequencing, and other molecular biology experiments, thus greatly improving sequencing quality. Attached Figure Description
[0051] Figure 1 is a schematic diagram of the pET28a carrier structure of Example 1.
[0052] Figure 2 shows the electrophoresis diagram during the Cy5-dA6P activity test. Embodiments of the present invention
[0053] The following are specific embodiments of the present invention, which further describe the technical solution of the present invention, but the scope of protection of the present invention is not limited thereto.
[0054] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.
[0055] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.
[0056] Unless otherwise specified, the solvent for all solutions or buffer solutions in the following examples is water.
[0057] In the quantitative experiments described below, three replicate experiments were conducted, and the average value of the results was taken.
[0058] The lysis buffer in the following examples is: 300 mmol / L NaCl, 50 mmol / L NaH2PO4, 10 mmol / L imidazole, pH=8.0.
[0059] The washing buffer in the following examples consists of 300 mmol / L NaCl, 50 mmol / L NaH2PO4, 20 mmol / L imidazole, and pH=8.0.
[0060] The elution buffer in the following examples is: 300 mmol / L NaCl, 50 mmol / L NaH2PO4, 250 mmol / L imidazole, pH=8.0.
[0061] The protein storage buffer used in the following examples is: 10 mmol / L Tris-HCl, 100 mmol / L KCl, 1 mmol / L LTT, 0.1 mmol / L EDTA, 0.5% Tween® 20, 0.5% IGEPAL® CA-630, 50% glycerol, pH=7.4.
[0062] The 10X phi29 reaction buffer in the following examples consists of: 500 mM Tris-HCl (pH 7.5 at 25°C), 100 mM MgCl2, 100 mM (NH4)2SO4, and 40 mM DTT.
[0063] The pET28-phi29 plasmid in the following examples: Based on the wild-type amino acid sequence SEQ ID NO.54 of Phi29 DNA polymerase and the codon preference of E. coli, the wild-type nucleic acid sequence SEQ ID NO.52 was synthesized and inserted between the NcoI and XhoI sites of the pET28a vector shown in Figure 1, without retaining the C-terminal His tag, to obtain the recombinant plasmid.
[0064] Template chains in the following embodiments:
[0065] 5'-AAAAAATCAAGGCTGGTCGGTCAGTC-3'.
[0066] The primer chains in the following examples:
[0067] 3'-GTTCCGACCAGCCAGTCAG-5'.
[0068] Cy3 is attached to the first thymine at the 3' end of the primer chain, and the O in the phosphodiester bond between the first and second thymines and the first G at the 3' end of the primer chain is replaced by S (site thiolation modification).
[0069] The Cy5-dA6P used in the following examples is a Cy5-labeled nucleotide substrate, synthesized by Anxuyuan Biotechnology Co., Ltd. The structural formula of Cy5-dA6P is as follows:
[0070] .
[0071] The 72 RCA Primer in the following examples is: CTGACTGCATCTAGACGTGACTGA.
[0072] The 72 Ad ssDNA in the following examples: synthesized by Aikerui Biotechnology Co., Ltd., is a 72nt single-stranded circular DNA with no fixed sequence.
[0073] In the examples, the amino acid sequences of the phi29 nucleic acid sequences expressed by SEQ ID NO.1-51 and the amino acid sequences of the phi29 DNA polymerase mutants shown by SEQ ID NO.54-104 correspond one-to-one.
[0074] The amino acid sequences shown in SEQ ID NO.105-106 are, in order, the amino acid sequences of the wild-type phi29 DNA polymerase with and without tags. Example 1
[0075] Construction of the original expression plasmids for S1 and Phi29 DNA polymerases:
[0076] Using pET28-phi29 plasmid as a template, the phi29 nucleic acid sequence shown in SEQ ID NO. 52 was amplified with high-fidelity polymerase. Then, the pET28-phi29 plasmid and the phi29 nucleic acid sequence shown in SEQ ID NO. 52 were digested with restriction endonucleases NcoI and XhoI. The digested plasmid fragments and the sequence were spliced together using T4 DNA ligase to obtain the nucleic acid sequence shown in SEQ ID NO. 53. The splicing product was transformed into E. coil DH5α competent cells for verification to obtain the original expression plasmid of Phi29 DNA polymerase.
[0077] Construction of plasmids for S2 and Phi29 DNA polymerase mutants:
[0078] Using partially overlapping primers (containing mutation sites) as shown in Table 1, the phi29 nucleic acid sequence shown in SEQ ID NO. 1-51 was amplified using the original expression plasmid of phi29 DNA polymerase as a template. The amplification reaction system (50 μL) consisted of: 10 μL 5x Pfu Reaction Buffer, 4 μL dNTP Mix (2.5 mM), 30 ng pET28-Phi29 plasmid, 1 μL Pfu DNA Polymerase, 1 μL forward primer (10 μM), and 1 μL reverse primer (10 μM). The amplification conditions were: 95℃ for 2 min, 30 cycles for [95℃ 20 s, 55℃ 20 s, 72℃ 7 min], 72℃ for 5 min, and 16℃∞. After being digested with Dpnl, the amplified product was transformed into E. coil DH5α competent cells, plated on LB plates containing kanamycin, and cultured at 37°C. Single colonies were then picked, plasmids were extracted, and sequenced to verify whether the phi29 DNA polymerase mutant plasmid was successfully constructed. The phi29 DNA polymerase mutant plasmid was obtained.
[0079] Table 1: Partially Overlapping Primers
[0080]
[0081]
[0082] Expression and purification of S3 and Phi29 DNA polymerase mutant proteins:
[0083] The validated original plasmid and mutant plasmid of Phi29 DNA polymerase were transformed into the expression host bacterium E. coil BL21(DE3) for further induction and purification. Due to the large number of mutant proteins, His-tagged protein purification beads were used for high-throughput purification of the mutant proteins.
[0084] 1. Induced expression of proteins
[0085] (1) Pick a single colony and inoculate it into LB liquid medium containing 100ug / mL Kana resistance, and incubate overnight at 37°C to activate it;
[0086] (2) Take the activated bacterial solution and inoculate 1% into 100ml of LB liquid medium containing 100ug / mL Kana resistance. Incubate at 37℃ and 200 r / min for about 2h until the logarithmic growth phase. The bacterial OD600 is about 0.6.
[0087] (3) Add IPTG to the culture medium to a final concentration of 0.2 mmol / L, and incubate overnight at 16℃ and 200 r / min for 8 h;
[0088] (4) Centrifuge at 4000 r / min and 4℃ for 5 min, and collect the bacterial cells;
[0089] (5) Discard the supernatant and resuspend the bacterial cells in 20 mL of Lysis buffer;
[0090] (6) Add PMSF to each tube of bacterial cells at a ratio of 1 wt%, and sonicate under ice bath conditions. Sonicate for 5 seconds with a 7-second interval, for a total of 99 times. The bacterial concentration difference after each collection is considered, and the bacterial solution becomes clear as the termination criterion.
[0091] (7) Add PEI to a final concentration of 0.3% and stir slowly for 10 min to remove DNA from the extract;
[0092] (8) Centrifuge at 18000 r / min for 40 min and collect the supernatant;
[0093] (9) SDS-PAGE gel electrophoresis was used to analyze protein expression. If the target protein was distributed in the supernatant, subsequent purification operations were performed.
[0094] 2. Purification of the target protein
[0095] (1) Vibrate the Ni-Charged MagBeads thoroughly to mix them;
[0096] (2) Take an appropriate amount of Ni-Charged MagBeads into a centrifuge tube and place the centrifuge tube on a magnetic separator to collect the magnetic beads;
[0097] (3) Add 1 mL of Lysis buffer to (2) and invert the tube several times to mix. Collect the magnetic beads using a magnetic separator and discard the supernatant. Repeat this step twice;
[0098] (4) Add the cell lysate containing the multihistidine-labeled protein prepared in step 1 to the test tube and gently invert the test tube to mix;
[0099] (5) Oscillate at a lower temperature for 60 minutes;
[0100] (6) Collect the magnetic beads using a magnetic separator and discard the supernatant;
[0101] (7) Add 1 mL of washing buffer to (6), stir well, collect the magnetic beads with a magnetic separator, discard the supernatant, and repeat twice;
[0102] (8) Add 500uL of Elution buffer to (7), mix well, and incubate in an ice bath for 5 min. You can wash repeatedly to increase the yield of the target protein.
[0103] (9) Collect the magnetic beads using a magnetic separator and transfer the supernatant containing the eluted protein into a clean test tube;
[0104] (10) Inject the supernatant containing the eluted protein from (9) into an ultrafiltration tube, centrifuge at 3000xg, replace and concentrate the protein storage buffer, and store at -20℃.
[0105] (11) SDS-PAGE gel electrophoresis was used to analyze the protein purification status. If there was a single target band with the correct molecular weight, the purification was successful and the amino acid sequence of the phi29 DNA polymerase mutant, as shown in SEQ ID NO.54 to SEQ ID NO.104, was obtained.
[0106] Cy5-dA6P activity test:
[0107] Rolling circle amplification was performed using 72 Ad ssDNA as a template. 10 μM 72 Ad ssDNA and 10 μM 72 RCA Primer were mixed and incubated at 95 °C for 3 min, followed by annealing on ice for 5 min to form the template-primer complex. The template-primer complex, dNTPs (100 nM each of Cy5-dA6P, dTTP, dCTP, and dGTP), 10X phi29 reaction buffer, and commercial phi29 DNA polymerase (1 μM) were incubated at 30 °C for 30 min. The reaction was terminated by adding 0.5 M EDTA; the negative control was without Cy5-dA6P. The sample was mixed with 6x DNA loading buffer and electrophoresed slowly at 100 V for 50 min on a 1% agarose gel. As shown in Figure 2, gel imaging revealed that a large number of long nucleic acid fragments were formed under the above reaction system and conditions. The marker ranges from 100 bp to 5000 bp, and the product obtained by adding Cy5-dA6P to lane 1. Cy5-dC6P can serve as a substrate for DNA elongation reactions.
[0108] Polymerization rate assay of wild-type and mutant Phi29 DNA polymerase:
[0109] One sample of System 1 and one sample of System 2 were combined as the first group for reaction and detection:
[0110] Following standard operating procedures, one part of system 1 was injected into syringe 1 of the stop-flow spectrometer (an SX20 stop-flow spectrometer from Applied Photophysics, UK), and one part of system 2 was injected into syringe 2 of the stop-flow spectrometer. The stop-flow spectrometer was used, and a single mixing (reactants mixed at a 1:1 ratio) was employed before the reaction. The change in Cy5 fluorescence intensity was detected to determine the rate of change in fluorescence intensity of the first group of Cy5-dC6P. The reaction conditions were a circulating water bath at 30°C for 5 minutes. The stop-flow spectrometer settings were: Bandwidth, 10 nm; Pathlength, 2 mm; Excitation slit size, 2 nm; Excitation wavelength for Cy3 was 550 nm; and emission wavelength for Cy3 was selected using a 665 nm cutoff filter.
[0111] Table 2 Reaction System
[0112]
[0113] Table 3: Polymerization rate test results of wild-type and mutant Phi29 DNA polymerases
[0114]
[0115] Set the Trigger to External, set the Repeat count, and click Acquire to obtain the data graph. Drag the data curves from the three measurements onto the same coordinate graph, click Trace, select Selection Dialog, and a processing interface will pop up. Select all curves, click Average to generate Average:0, select the averaged curve, click Smooth, select the number of smoothing cycles, and generate Smooth(s) as the smoothed curve. Select the smoothed curve, click Fit, and select the single exponential model a.exp (-kx) +c, set the Fit Range, then click Estimate, Fit to generate Fit(f) as the fitted curve. The fluorescence rate constant k is obtained; the larger the k value, the slower the polymerization rate. The final results are shown in Table 3. The fluorescence rate constant of the wild-type Phi29 DNA polymerase (WT) is 0.16 ± 0.0002, while the others are mutant Phi29 DNA polymerases with amino acid sequences as shown in Example 1 of this invention (SEQ ID NO. 54-104). A total of 51 mutation sites with fluorescence rate constants higher than WT were screened. This shows that the polymerization rate of the mutants of this invention is reduced to varying degrees compared to the wild-type Phi29 DNA polymerase (WT), successfully solving the problem of excessively fast polymerization rate caused by Phi29 DNA polymerase in practical applications. This improves its accuracy, stability, and reliability in DNA amplification, sequencing, and other molecular biology experiments, greatly enhancing sequencing quality.
[0116] The embodiments herein cover any points not exhaustively within the scope of the technical claims of this invention, as well as new technical solutions formed by equivalent substitutions of one or more technical features in the embodiments. These are all within the scope of the claims of this invention. Furthermore, in all listed or unlisted embodiments of this invention, each parameter in the same embodiment merely represents an instance (i.e., a feasible solution), and there is no strict coordination or limitation relationship between the parameters. The parameters can be substituted for each other without violating axioms and the claims of this invention, unless otherwise stated.
[0117] The technical means disclosed in this invention are not limited to those described above, but also include technical solutions composed of any combination of the above technical features. The above descriptions are specific embodiments of this invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications are also considered within the scope of protection of this invention.
[0118] The specific embodiments described herein are merely illustrative of the spirit of the invention. Those skilled in the art to which this invention pertains may make various modifications or additions to the described specific embodiments or use similar methods to substitute them, without departing from the spirit of the invention or exceeding the scope defined by the appended claims.
Claims
1. A phi29 DNA polymerase mutant with reduced polymerization rate, characterized in that, The phi29 DNA polymerase mutant includes any one of A1) to A2): A1) An amino acid sequence as shown in any one of SEQ ID NO. 54 to SEQ ID NO. 104; A2) An amino acid sequence obtained by attaching a tag to the middle and / or N-terminus and / or C-terminus of the amino acid sequence shown in A1).
2. The phi29 DNA polymerase mutant with reduced polymerization rate according to claim 1, characterized in that, The tags include at least one of those that facilitate the dissolution, expression, purification, fixation, and detection of the phi29 DNA polymerase mutant.
3. A biomaterial related to the phi29 DNA polymerase mutant of claim 1, characterized in that, The biomaterial is any one of B1) to B4): B1) A nucleic acid molecule encoding the phi29 DNA polymerase mutant of claim 1; B2), an expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1) or the expression cassette described in B2); B4) Recombinant biological cells containing the nucleic acid molecule described in B1), the expression cassette described in B2), or the recombinant vector described in B3).
4. The biomaterial according to claim 3, characterized in that, The nucleotide sequence of the nucleic acid molecule is shown in any one of SEQ ID NO.1 to SEQ ID NO.
51.
5. The biomaterial according to claim 3, characterized in that, The expression cassette is DNA expressing the phi29 DNA polymerase mutant in host cells.
6. The biomaterial according to claim 3, characterized in that, The recombinant vector is a recombinant vector obtained by inserting a nucleic acid molecule encoding the phi29 DNA polymerase mutant into the multiple cloning site of a biological vector.
7. The biomaterial according to claim 6, characterized in that, Biological vectors are at least one of plasmids, granules, bacteriophages, and viral vectors.
8. The use of a primer combination in amplifying a nucleic acid molecule encoding the phi29 DNA polymerase mutant of claim 1.
9. An enzyme preparation, characterized in that, Includes the phi29 DNA polymerase mutant as described in claim 1.
10. A method for preparing the phi29 DNA polymerase mutant according to claim 1, characterized in that, The method includes the following steps: introducing the coding gene of the phi29 DNA polymerase mutant according to claim 1 into a biological cell, so that the coding gene is expressed, and obtaining the phi29 DNA polymerase mutant.
11. A method for amplifying or sequencing template DNA, characterized in that, The method includes the following steps: amplifying or sequencing template DNA using the phi29 DNA polymerase mutant as described in claim 1.
12. The method for amplifying or sequencing template DNA according to claim 11, characterized in that, The amplification or sequencing process involves a labeled nucleotide substrate, the structural formula of which is: 。 13. The application of any one of C1) to C3) in any one of D1) to D4), characterized in that, C1) to C3) and D1) to D4) are shown below: C1) The phi29 DNA polymerase mutant according to claim 1; C2), the biomaterial described in claim 3; C3), the enzyme preparation according to claim 9; D1) Nucleic acid amplification; D2) Preparation of nucleic acid amplification-related products; D3), sequencing; D4) Prepare sequencing-related products.
14. A nucleic acid amplification or sequencing related product, characterized in that, Includes the phi29 DNA polymerase mutant of claim 1 or the enzyme preparation of claim 9.
15. A nucleic acid amplification or sequencing related product according to claim 14, characterized in that, The product also includes non-natural substrates.