Phi29 dna polymerase mutant with improved amplification efficiency and application thereof

Abstract

The application belongs to the technical field of nucleic acid amplification, and discloses a Phi29 DNA polymerase mutant with improved amplification efficiency and application thereof. Compared with wild-type phi29 DNA polymerase, the Phi29 DNA polymerase mutant provided by the application comprises at least one of the following mutation sites: M102Q, P558L, T534R, M102R, T534P, Y224K, G217E, T140P, H284Y, V19P. The Phi29 DNA polymerase mutant has excellent amplification efficiency, and the band brightness obtained by amplification under the same conditions is significantly higher than that of the wild type, and the amplification efficiency is as high as more than twice that of the wild type. The application has good application prospect in the field of nucleic acid amplification and sequencing.

Description

Technical Field

[0001] This invention belongs to the field of nucleic acid amplification technology, specifically relating to a Phi29 DNA polymerase mutant with improved amplification efficiency and its application. Background Technology

[0002] Phi29 DNA polymerase is a type of enzyme derived from Bacillus subtilis (… Bacillus subtilis Phi29 DNA polymerase, a group B DNA polymerase, possesses a high continuous synthesis capacity, allowing it to extend DNA strands exceeding 70 kb with a single binding, far surpassing the 1-2 kb extensions of common polymerases (such as Taq polymerase). It exhibits strong strand displacement activity, driving DNA double-strand dissociation without helicase, making it suitable for rolling circle amplification (RCA) and whole genome amplification (WGA). Phi29 DNA polymerase demonstrates high fidelity, with a mismatch rate as low as 10% during amplification. -6 -10 -7 It is superior to most room-temperature polymerases.

[0003] With the development of synthetic biology, single-cell sequencing, and nanopore sequencing technologies, traditional Phi29 enzymes are gradually facing bottlenecks. Due to its poor thermostability (optimal temperature of 30℃), Phi29 DNA polymerase is incompatible with high-temperature pre-denaturation steps and is prone to introducing contaminants. The reaction rate of Phi29 DNA polymerase at 30℃ is only 20-30 nt / s, limiting its amplification speed and making it difficult to meet high-throughput requirements. Secondly, Phi29 DNA polymerase has poor adaptability to complex templates and exhibits low amplification efficiency in regions with high GC content or rich secondary structures. In scenarios involving long fragment amplification, this Phi29 DNA polymerase is prone to premature dissociation, resulting in short-fragment bias and uneven product length distribution.

[0004] Therefore, it is necessary to develop a novel, highly stable Phi29 DNA polymerase with good amplification performance. Summary of the Invention

[0005] To address the need for improved amplification efficiency and enhanced DNA polymerase thermostability in existing technologies, this invention provides a Phi29 DNA polymerase mutant with improved amplification efficiency and its applications. The specific technical solution is as follows: In a first aspect, a Phi29 DNA polymerase mutant with improved amplification efficiency is provided, which is obtained by single-point mutation or multi-point combination mutation of the amino acid sequence shown in SEQ ID NO. 1 at the following sites: M102Q, P558L, T534R, M102R, T534P, Y224K, G217E, T140P, H284Y, V19P.

[0006] Secondly, the present invention provides a coding gene for the above-mentioned Phi29 DNA polymerase mutant.

[0007] Thirdly, the present invention provides an expression vector containing the above-mentioned coding gene.

[0008] Furthermore, the expression vector is a plasmid, bacteriophage, or viral vector.

[0009] Fourthly, the present invention provides a host cell comprising the above-mentioned Phi29 DNA polymerase mutant or the above-mentioned encoding gene.

[0010] Furthermore, the host cell is *Escherichia coli* (E. coli). Escherichia coli ).

[0011] Fifthly, this invention provides the application of a Phi29 DNA polymerase mutant in nucleic acid amplification. The Phi29 DNA polymerase mutant of this invention, compared to the wild-type phi29 DNA polymerase, produces bands with significantly higher brightness under the same conditions. Therefore, the Phi29 DNA polymerase mutant of this invention exhibits excellent amplification efficiency.

[0012] In a sixth aspect, the present invention provides a kit for nucleic acid amplification comprising the above-mentioned Phi29 DNA polymerase mutant.

[0013] In a seventh aspect, the present invention provides a method for amplifying DNA molecules, comprising the following steps: The Phi29 DNA polymerase mutant was used to amplify DNA molecules.

[0014] Eighthly, the present invention provides an application of the Phi29 DNA polymerase mutant in gene sequencing.

[0015] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a Phi29 DNA polymerase mutant with enhanced amplification efficiency, obtained by single-point or multi-point combination mutations at the following sites in the amino acid sequence shown in SEQ ID NO.1: M102Q, P558L, T534R, M102R, T534P, Y224K, G217E, T140P, H284Y, and V19P. Compared to wild-type phi29 DNA polymerase, the above-mentioned Phi29 DNA polymerase mutant exhibits higher enzyme activity and higher amplification efficiency, up to more than twice that of the wild-type. The above-mentioned Phi29 DNA polymerase mutant shows great promise for applications in nucleic acid amplification and gene sequencing. Attached Figure Description

[0016] Figure 1 The figure shows the expression and purification results of wild-type Phi29 DNA polymerase. M in the figure is the protein marker, and lanes 1-7 represent different mutants. Figure 2 The figure shows the results of multiple strand substitution amplification. M is the DNA marker, 1 is the enzyme-free blank control, 2 is the amplification result of wild-type Phi29 DNA polymerase, and 3-7 are the amplification results of Phi29 DNA polymerase mutant. Figure 3 The image shows the results of rolling circle amplification. In the image, M is the DNA marker, 1 is the enzyme-free blank control, 2 is the amplification result of wild-type Phi29 DNA polymerase, and 3-12 are the amplification results of Phi29 DNA polymerase mutant.

[0017] Figure 4 The image shows the results of rolling circle amplification. In the image, M is the DNA marker, 1 is the enzyme-free blank control, 2 is the amplification result of wild-type Phi29 DNA polymerase, and 3-9 are the amplification results of Phi29 DNA polymerase mutant. Detailed Implementation

[0018] To enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. It should be noted that the following detailed descriptions are exemplary and are only some embodiments of the present invention, not all embodiments.

[0019] Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0020] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. The experimental materials used in the embodiments of this invention are all conventional experimental materials in the art and are commercially available. Experimental methods not specifying detailed conditions were performed according to conventional experimental methods or the supplier's recommended operating instructions. The sequences synthesized in the embodiments of this invention were synthesized by Wuhan Jinkairui Biotechnology Co., Ltd.

[0021] In the following examples, the *10×PCR Buffer (pH 7.5) used consisted of: 500 mM Tris-HCl, 100 mM MgCl2, 100 mM (NH4)2SO4, and 40 mM DTT.

[0022] In the following examples, the pET 28a vector was purchased from Invitrogen, the restriction endonuclease was purchased from Sangon Biotech (Shanghai) Co., Ltd., and the host cell Escherichia coli BL21(DE3) was purchased from Invitrogen.

[0023] In the following examples, the nucleotide sequence of the wild-type Phi29 DNA polymerase is shown in SEQ ID NO. 2, and the nucleotide sequence was synthesized by Wuhan Jinkairui Biotechnology Co., Ltd. The amino acid sequence of the Phi29 DNA polymerase is shown in SEQ ID NO. 1.

[0024] In the following examples, site-directed mutagenesis was performed on the wild-type Phi29 DNA polymerase, specifically by mutagenesis at the following sites in the amino acid sequence shown in SEQ ID NO.1: M102Q, P558L, T534R, M102R, T534P, A531K, M102K, Q497P, R187G, Q497A, Y224K, G217E, T140P, S194A, H284Y, V19P, and W277Y. The forward and reverse primers used to construct the mutants in the following examples are shown in Table 1.

[0025] Table 1

[0026] Example 1 This embodiment constructs the Phi29 DNA polymerase mutant expression vector according to the following steps: (1) Construction of wild-type expression vector A histidine tag (HHHHHHHHH) was designed at the 3' end of the wild-type Phi29 DNA polymerase. pET28a(+) was selected as the plasmid vector, and Nde I and Xhol I were used as restriction enzyme sites for recombination to obtain the wild-type recombinant plasmid pET-28a / phi29 WT. The obtained wild-type recombinant plasmid was transformed into DH5α competent cells, and positive single clones were screened for sequencing verification. The verification results were consistent with expectations.

[0027] (2) Construction of mutant expression vectors Single colonies of the wild-type plasmid obtained in the above steps were picked and activated in 5 mL LB medium containing 50 μg / mL kanamycin. Wild-type plasmids were extracted using a plasmid extraction kit. Using the extracted plasmids as templates, forward and reverse primers for site-directed mutagenesis were designed using Snapgene software (as shown in Table 1) and synthesized by Jinjin Kairui Biotechnology Co., Ltd. Site-directed mutagenesis PCR was performed according to the reaction system shown in Table 2 and the PCR amplification program shown in Table 3. The obtained products were then digested with 1 μL DpnI enzyme to degrade the original template, yielding mutant linear plasmids. The plasmid was transformed into DH5α competent cells, and positive single clones were screened and sequenced for verification. The results were consistent with expectations, yielding recombinant expression plasmids for single-point mutants M102Q, P558L, T534R, M102R, T534P, A531K, M102K, Q497P, R187G, Q497A, Y224K, G217E, T140P, S194A, H284Y, V19P, and W277Y, as well as V1... Recombinant expression plasmids of mutants with the following mutations: 9P+M102R double-point combination mutation, V19P+M102Q double-point combination mutation, V19P+T534P double-point combination mutation, T140P+M102R double-point combination mutation, T140P+M102Q double-point combination mutation, T140P+T534P double-point combination mutation, W277Y+M102R double-point combination mutation, W277Y+M102Q double-point combination mutation, and W277Y+T534P double-point combination mutation.

[0028] The correctly sequenced mutant plasmid and wild-type were transformed into BL21 competent cells, plated on LB agar medium containing 50 μg / mL kanamycin, and positive single colonies were screened for subsequent mutant protein expression.

[0029] Table 2. Reaction systems for site-directed catastrophe

[0030] Table 3 PCR amplification program

[0031] Example 2 In this embodiment, the recombinant expression plasmids of wild type and mutant constructed in Example 1 were expressed, and the Phi29 DNA polymerase and its mutant obtained from the expression were purified separately according to the following steps: (1) Expression The E. coli BL21 positive monoclonal colonies obtained in Example 1 were picked and added to 5 mL of LB medium containing 50 μg / mL kanamycin. After activation at 37°C overnight, they were inoculated into 100 mL of LB medium containing 50 μg / mL kanamycin at a volume ratio of 1:100 and cultured in a shaker at 37°C until 0D. 600 The concentration was 0.8. IPTG was added to the bacterial culture to a final concentration of 1.0 mmol / L. The temperature was lowered to 16℃ and the culture was shaken for 16 h. The induced bacterial cells were collected by centrifugation and weighed. The wet weight of the bacterial cells was recorded and stored at -80℃.

[0032] (2) Purification After inducing bacterial expression, the cells were resuspended in 5 mL of lysis buffer (50 mM Tris-HCl, 300 mM NaCl, pH 7.8) per gram of wet weight of cells. The resulting cell suspension was then lysed using an ultrasonic disruptor. The sonication conditions were: 40% power, 3 seconds on, 6 seconds off, for a total disruption time of 40 min. The resulting lysate was centrifuged at 4°C and 12000 rpm for 10 min. The supernatant was transferred to a 50 mL centrifuge tube to obtain the protein solution to be purified; the precipitate was discarded.

[0033] After equilibrating the Ni-NTA column (purchased from Sangon Biotech (Shanghai) Co., Ltd.) with buffer A (50mM Tris-HCl, 300mM NaCl, 20mM imidazole, pH 7.8), the protein solution to be purified was loaded into the column. The flow-through buffer was then collected and used to prepare a 200mM imidazole solution with buffer B (50mM Tris-HCl, 1mM EDTA, 300mM NaCl, 1mM DTT, 500mM Mimidazole, pH 7.8) using buffer A for elution. The elution volume was 10 times the column volume. The eluent containing the target protein was collected using a protein ultrafiltration concentrator and centrifuged at 7500 rpm at 4°C. The ultrafiltration was repeated until the solution volume was 500 μL. 3 ml of enzyme storage buffer (composition: 10 mM Tris-HCl, 100 mM KCl, 0.1 mM EDTA, 1 mM DTT) was added to the protein ultrafiltration concentrator, and the mixture was centrifuged at 7500 rpm at 4°C for desalting. This process was repeated three times to obtain the wild-type enzyme and protein solutions of each mutant. SDS-PAGE protein electrophoresis confirmed that each mutant showed a clear band at approximately 68 kDa, consistent with the theoretical molecular weight, indicating successful expression of the target protein. For example, the electrophoresis results of the mutants are shown below. Figure 1As shown in the figure, M is the protein marker, and lanes 1-8 refer to the purified proteins of Y224K, A324V, G217E, T140P, S194A, H284Y, V19P, and W277Y, respectively.

[0034] Example 3 In this embodiment, the purified Phi29 DNA polymerase mutants obtained in Example 2 were applied to multiple strand displacement amplification to evaluate the amplification activity of each mutant enzyme.

[0035] This step uses a single-stranded circular plasmid template and random primers for multiple-strand displacement amplification. Catalyzed by Phi29 DNA polymerase, dNTPs are gradually incorporated, amplifying the single-stranded plasmid template to form double-stranded DNA products. Simultaneously, the strand displacement activity of Phi29 DNA polymerase breaks down the newly synthesized double strands into single strands, providing templates for the next round of amplification. The final products are single-stranded and double-stranded DNA of varying lengths. SuperGreen is a highly sensitive nucleic acid dye that binds to both double-stranded and single-stranded DNA. Upon binding to the amplification products, it produces a fluorescent signal, the intensity of which is positively correlated with the concentrations of dsDNA and ssDNA. This step utilizes SuperGreen to generate fluorescent signals by binding to the amplification products; measuring the fluorescence intensity over a certain time allows for comparison of DNA polymerase activity.

[0036] The steps are as follows: Add the components shown in Table 4 to centrifuge tubes, place them in a PCR instrument, incubate at 95 ℃ for 3 min, then immediately cool on ice for 5 min. Add 1 mg of the enzyme to be tested, vortex the mixture, and then place it in the PCR instrument for amplification at 30 ℃ for 3 h. Inactivate the enzyme at 65 ℃ for 10 min and stop the amplification reaction. Run the amplified products on agarose gel electrophoresis to evaluate the enzyme's amplification activity. The results are shown in Table 4. Figure 2 As shown. Figure 2 In the diagram, lane M represents the standard molecular weight of nucleic acid; lane 1 is the enzyme-free control; lanes 2-12 are, in order, wild type, T534P, V19P+M102R, V19P+M102Q, V19P+T534P, T140P+M102R, T140P+M102Q, T140P+T534P, W277Y+M102R, W277Y+M102Q, and W277Y+T534P.

[0037] Table 4 Enzyme activity detection reaction system

[0038] The test results showed that the bands in lanes 3, 6, 7, 9, 10, and 11 were very bright, significantly higher than the wild type; lane 8 was also brighter than the wild type, while the others were weaker or comparable. It is evident that the single-site mutant T534P and the two-site combined mutants V19P+T534P, T140P+M102R, T140P+T534P, W277Y+M102R, and W277Y+M102Q exhibited significantly increased enzyme activity compared to the wild type. Quantitative fluorescence analysis showed that the W277Y+M102Q combined mutant exhibited the best enzyme activity, at 306.7% of the wild type. Furthermore, the T140P+M102R and W277Y+M102R combined mutants also showed extremely high enzyme activity, approximately 303.8% and 304.6% of the wild type, respectively.

[0039] Example 4 This embodiment describes the rolling circle amplification detection of the obtained Phi29 DNA polymerase mutant and wild type.

[0040] Based on the principle of rolling circle amplification (RCA), this embodiment uses a single-stranded circular plasmid template and thiolated specific primers for rolling circle amplification. Under the catalysis of DNA polymerase, dNTPs are gradually incorporated, and the single-stranded plasmid template is amplified to form a double-stranded DNA product. Simultaneously, the strand displacement activity of Phi29 DNA polymerase dissociates the newly synthesized double strand into single strands, providing a template for the next round of amplification. The final product is a long single-stranded DNA containing repetitive sequences. SuperGreen is a highly sensitive nucleic acid dye that can bind to both double-stranded and single-stranded DNA. After binding to the amplification product, it generates a fluorescent signal, and the intensity of the fluorescent signal is positively correlated with the concentrations of dsDNA and ssDNA. In this step, the fluorescence signal generated by SuperGreen binding to the amplification product can be compared by measuring the fluorescence signal intensity over a certain period of time.

[0041] The specific steps are as follows: Add the components shown in Table 5 to centrifuge tubes, place them in a PCR instrument, incubate at 95 ℃ for 5 min, then immediately cool on ice for 5 min. Add 1 mg of the enzyme to be tested, vortex the mixture, centrifuge, and then place it in a PCR instrument for amplification at 30 ℃ for 3 h. Inactivate the enzyme at 65 ℃ for 10 min and stop the amplification reaction. Run the amplified products on an agarose gel electrophoresis gel to evaluate the enzyme amplification activity. The results are shown in Table 5. Figure 3 and Figure 4 As shown. Figure 3In the diagram, lane M represents the standard molecular weight of nucleic acid; lane 1 is the enzyme-free control; lanes 2-12 are wild type, M102Q, P558L, T534R, M102R, T534P, A531K, M102K, Q497P, R187G, and Q497A, respectively. Figure 4 In the diagram, lane M represents the standard molecular weight of nucleic acid; lane 1 is the enzyme-free control; lanes 2-9 represent wild type, Y224K, G217E, T140P, S194A, H284Y, V19P, and W277Y, respectively.

[0042] Table 5 Enzyme activity detection reaction system

[0043] The test results showed that the fluorescence signal intensity of mutants M102Q, P558L, T534R, M102R, T534P, Y224K, G217E, T140P, H284Y, and V19P after amplification was significantly higher than that of the wild type. Quantitative fluorescence analysis revealed that the enzyme activities of the mutants T534P, M102R, M102Q, T140P, V19P, and W277Y were 295.1%, 262.5%, 248.0%, 289.0%, 278.5%, and 209.8% of the wild type, respectively, with each mutant exhibiting more than twice the intensity of the wild type. Therefore, the above single-point mutants M102Q, P558L, T534R, M102R, T534P, Y224K, G217E, T140P, H284Y, and V19P exhibit significantly increased enzyme activity compared to the wild type.

Claims

1. A Phi29 DNA polymerase mutant with improved amplification efficiency, characterized in that: Obtained by single-point or multi-point combination mutations at the following sites from the amino acid sequence shown in SEQ ID NO.1: M102Q, P558L, T534R, M102R, T534P, Y224K, G217E, T140P, H284Y, V19P.

2. The encoding gene of the Phi29 DNA polymerase mutant as described in claim 1.

3. An expression vector comprising the encoding gene of claim 2.

4. The expression vector as described in claim 3, characterized in that: The expression vector is a plasmid, bacteriophage, or viral vector.

5. A host cell comprising the coding gene of claim 2 or the expression vector of claim 3.

6. The host cell as described in claim 5, characterized in that: The host cell is Escherichia coli.

7. The application of the Phi29 DNA polymerase mutant as described in claim 1 in nucleic acid amplification.

8. A reagent kit for nucleic acid amplification, characterized in that: It includes the Phi29 DNA polymerase mutant as described in claim 1.

9. A method for amplifying DNA molecules, characterized in that: Includes the following steps: DNA molecules were amplified using the Phi29 DNA polymerase mutant as described in claim 1.

10. The application of the Phi29 DNA polymerase mutant as described in claim 1 in gene sequencing.

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