Mutant Taq DNA polymerase for rapid PCR and application thereof

Abstract

The invention discloses a mutant Taq DNA polymerase for rapid PCR and application thereof, and belongs to the technical field of enzyme engineering, the amino acid sequence of the mutant Taq DNA polymerase corresponds to the sequence as shown in SEQ ID NO: 4, and the mutant Taq DNA polymerase has a plurality of mutation sites as follows: D58E, K292N, G304A, E507K and E742K; compared with a wild Taq DNA polymerase, the mutant Taq DNA polymerase provided by the invention has the advantages that the amplification rate is increased by more than 4 times, the rapid amplification performance is excellent, the PCR inhibitor resistance and the thermal stability are excellent, and meanwhile, the mutant Taq DNA polymerase is matched with a rapid PCR buffer solution provided by the invention, so that the amplification extension of 40 PCR cycles can be completed within the net duration of 350sec, the amplification of 40 cycles can be completed within 19min, and the application prospect is wide. The PCR detection time is remarkably shortened, and the requirement of rapid PCR detection is met.

Description

Technical Field

[0001] The present invention relates to the technical field of enzyme engineering, and particularly relates to a mutant Taq DNA polymerase.

[0002] The present invention also relates to a rapid PCR amplification kit.

[0003] The present invention also relates to a method for rapid PCR amplification. Background Art

[0004] The emergence of heat-resistant DNA polymerase in 1988 made PCR automation a reality. With the development of technology and increasingly stringent application requirements, the TaqMan probe-based qPCR technology has been widely used in clinical diagnosis, food safety detection, environmental monitoring and other fields because it can achieve completely closed-tube operation, avoiding the aerosol contamination problem caused by opening the lid in traditional PCR, and can also achieve qualitative and quantitative analysis, as well as having the advantages of high throughput, high sensitivity and high efficiency. However, the TaqMan probe-based qPCR technology usually requires nucleic acid extraction of the sample to be detected and then PCR amplification detection. However, problems such as nucleic acid loss and cross-contamination between samples are likely to occur during the nucleic acid extraction process, and the extraction steps are relatively cumbersome, and the overall detection process takes a long time.

[0005] With the development of molecular diagnostic technologies, rapid diagnosis has gradually become the demand and goal of the development of the molecular diagnostic technology field. Especially in recent years, with the prevalence of global infectious diseases, the demand for rapid nucleic acid detection has increased sharply. Compared with conventional qPCR technology, one of the rapid qPCR modes is to achieve rapid diagnosis by shortening the annealing extension time or combining microfluidic technology. Specifically, "rapid PCR" or "rapid cycling" generally refers to completing 30 cycles within 10 - 60 minutes. The actual time for each cycle is more than the sum of the times usually programmed for denaturation, annealing and extension, because time is required for the temperature change between each of these stages. Over the years, the system has become faster, and the kinetic requirements for denaturation, annealing and extension have become clearer. And one of the core technologies to achieve this technology depends on highly efficient reaction enzymes. Taq DNA polymerase is a thermostable DNA polymerase isolated from the thermophilic bacterium Thermus aquaticus. Its optimal catalytic temperature is 70 - 75°C, and its half-life at 95°C is 45 - 50 min. Its good heat resistance characteristics have greatly promoted the specificity, automation and amplification efficiency of PCR. However, the wild-type Taq DNA polymerase has low activity, insufficient extension performance, weak stability, poor inhibition resistance and low fidelity. Therefore, obtaining high-performance Taq DNA polymerase through genetic engineering technology is of great significance for the development of rapid qPCR technology.

[0006] Currently, there are several ways to modify the Taq DNA polymerase molecule as follows:

[0007] (1) Deletion modification: Studies have shown that deleting the 5'-3' exonuclease domain of Taq DNA polymerase can improve the thermal stability, fidelity, and salt ion tolerance of Taq DNA polymerase.

[0008] (2) Domain recombination or fusion: Chimerizing or fusing an exogenous domain or protein with known properties to a DNA polymerase can provide protein modifications achieved by point mutations or small sequence insertions / deletions, and modified variants with target properties can be obtained quickly and effectively.

[0009] (3) Directed evolution: Directed evolution of Taq DNA polymerase usually uses site-directed mutagenesis methods and methods of directed screening in a random mutation library. Site-directed mutagenesis: By methods such as polymerase chain reaction, desired changes are introduced into the target DNA fragment. The target DNA fragment can be a genome or a plasmid. Desired changes, usually changes representing favorable directions, including base addition, deletion, point mutation, etc., are introduced into the target DNA fragment, which can quickly and efficiently improve the traits and characteristics of the target protein expressed by the DNA. Directed screening of a random mutation library: Based on a constructed large mutant library and combined with high-throughput screening methods, mutants with high performance can be effectively obtained after multiple rounds of screening. In 2001, Ghadessy et al. proposed a compartmentalized self-replication technique (CSR) to screen mutants with target properties. This technique consists of a simple feedback loop that involves a polymerase that only replicates its own coding gene, i.e., self-replication. Self-replication occurs in discrete, spatially separated, and unconnected compartments formed by a thermostable water-in-oil emulsion. Compartmentalization ensures the connection between phenotype and genotype, that is, it ensures that each polymerase only replicates its own coding gene and excludes the coding genes in other compartments. As a result, the adaptive gain of the polymerase is directly and proportionally converted into genetic amplification of the gene encoding the polymerase.

[0010] Currently, the reported modified Taq DNA polymerase has the following improved performance characteristics, such as thermal stability, sensitivity, reverse transcription activity, tolerance to blood and fluorescent dyes, amplification of low-template substances, and long fragments, etc. For example, Patent No. CN116497001 discloses a Taq DNA polymerase mutant. Based on the method of emulsion PCR (ePCR), two mutants, Y686R / E687K / Q690G and Y686R / E687K / Q690V, containing the following mutation sites were screened out. The amplification effect of the obtained mutants was that they could amplify an amplification fragment of 2 kb, and their sensitivity was that they could amplify for 10 -3ng / μL and 10 -4 For the target fragment with a concentration of 1 ng / μL to 10 ng / μL, the net extension time of PCR amplification exceeds 700 sec. Although the performance is improved compared with the unmodified wild-type Taq DNA polymerase, the amplification effect and amplification time of the above mutants still need to be further improved for the field of rapid PCR applications. Patent No. CN118006582A discloses a Taq DNA polymerase mutant, which obtains 4 mutants through CSR technology. The net extension time of the best mutant in terms of rapid PCR ability is 900 sec, far exceeding the fastest reported PCR amplification extension time of 620 sec, and does not disclose the inhibitor tolerance and sensitivity of the Taq DNA polymerase mutant. Another example is that Patent No. CN117683741A discloses a Taq DNA polymerase mutant and its application, which screens out 30 Taq DNA polymerase mutants through error-prone PCR to construct a random mutation library. Compared with the wild-type Taq DNA polymerase, its specific activity is increased by 2-5 times, and the amplification rate is increased by more than 2 times. The net extension time of the best mutant M5 in qPCR is 1980 sec, indicating that there is still a large room for improvement in shortening the detection time of this mutant.

[0011] In summary, although there are currently many studies on the performance improvement of Taq DNA polymerase, it still cannot meet the requirements of short detection time, high amplification efficiency of DNA polymerase, high detection sensitivity, and inhibitor tolerance in current rapid PCR. In view of this, the present invention is specifically proposed. Summary of the Invention

[0012] The purpose of the present invention is to provide a mutant Taq DNA polymerase for rapid PCR and its application in view of the above problems. The mutant Taq DNA polymerase of the present invention has a high amplification rate and excellent tolerance to whole blood, plasma, serum, SDS, porcine bile salt, ethanol, and M-MLV reverse transcriptase, and is suitable for rapid PCR, significantly reducing the time of PCR detection.

[0013] The technical solution adopted by the present invention is as follows:

[0014] A mutant Taq DNA polymerase, the amino acid sequence of the mutant Taq DNA polymerase corresponds to the sequence shown in SEQ ID NO: 4, and has the following multiple mutation sites: D58E, K292N, G304A, E507K, and E742K.

[0015] Furthermore, the amplification rate of the mutant Taq DNA polymerase is at least 1.5 times that of the wild-type Taq DNA polymerase; preferably at least 2 times; preferably at least 3 times; preferably at least 4 times.

[0016] A polynucleotide molecule, the sequence of the polynucleotide molecule corresponding to the nucleotide sequence shown in SEQ ID NO:3, and the polynucleotide molecule encodes the mutant Taq DNA polymerase.

[0017] A recombinant vector, the vector containing the polynucleotide molecule.

[0018] A host cell, the host cell comprising the vector or the polynucleotide molecule chromosomally integrated.

[0019] A rapid PCR amplification kit, the kit comprising the mutant Taq DNA polymerase.

[0020] Further, the kit further includes a 5×Fast buffer, and the pH value of the 5×Fast buffer is 9.0-9.6;

[0021] The 5×Fast buffer includes a buffer pair, a monovalent cation, an enhancer, and a surfactant.

[0022] Further, the buffer pair is Tris-HCl;

[0023] The monovalent cation includes NH4 + , K + One of them;

[0024] The enhancer includes BSA and TMAC;

[0025] The surfactant includes any one of Tween 20, CA-630, and Triton X-100.

[0026] Further, the kit further includes at least one of a dNTP mixture, a primer pair, Mg 2+ and water.

[0027] A method for rapid PCR amplification, the method comprising performing PCR amplification using the 5×Fast buffer in the kit and the mutant Taq DNA polymerase.

[0028] In summary, due to the adoption of the above technical solutions, the beneficial effects of the present invention are:

[0029] The mutant Taq DNA polymerase of the present invention containing the mutation sites of D58E, K292N, G304A, E507K and E742K not only has an amplification rate increased by more than 4 times compared with the wild-type Taq DNA polymerase, excellent rapid amplification performance, but also has excellent tolerance to whole blood, plasma, serum, SDS, porcine bile salt, ethanol and M-MLV reverse transcriptase.

[0030] The mutant Taq DNA polymerase of the present invention in combination with the 5×Fast buffer designed by the present invention can achieve rapid PCR amplification, and can complete 40 cycles of PCR amplification extension within a net duration of 350 seconds, realizing the amplification of 40 cycles in 19 minutes, significantly reducing the PCR detection duration and meeting the requirements of rapid PCR detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is a state diagram after the amplification of the emulsion PCR system in Example 1 of the present invention;

[0032] Figure 2 It is an agarose gel electrophoresis diagram of the mutant second pressure screening amplification in Example 1 of the present invention;

[0033] Figure 3 It is a comparison result diagram of the Mut 52 Taq DNA polymerase gene sequence and the wild-type Taq DNA polymerase gene sequence in Example 1 of the present invention;

[0034] Figure 4 It is an SDS-PAGE electrophoresis diagram of the purified Mut52 Taq DNA polymerase in Example 2 of the present invention;

[0035] Figure 5 It is an agarose gel electrophoresis comparison diagram of the amplification rate detection of Mut52 Taq DNA polymerase and wild-type Taq DNA polymerase in Example 3 of the present invention;

[0036] Figure 6 A is the heat resistance test result of the wild-type Taq DNA polymerase in Example 5 of the present invention, Figure 6 B is the heat resistance test result of Mut52 Taq DNA polymerase;

[0037] Figure 7 A is the amplification result of Mut52 Taq DNA polymerase for the linear gradient template based on the rapid detection program 1 in Example 7 of the present invention, Figure 7 B is the amplification result of Mut52 Taq DNA polymerase for the linear gradient template based on the rapid detection program 7, Figure 7C is the amplification result of Mut52 Taq DNA polymerase for a linear gradient template using 5×Fast buffer that has been freeze-thawed 20 times based on Quick Detection Procedure VII. Detailed implementation mode

[0038] The present invention will be described in detail below in conjunction with the accompanying drawings.

[0039] In order to make the purpose, technical solution and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0040] The present invention provides a mutant Taq DNA polymerase, also known as a hot start fast Taq DNA polymerase. The amino acid sequence of the mutant Taq DNA polymerase corresponds to the sequence shown in SEQ ID NO:4 and has the following multiple mutation sites: D58E, K292N, G304A, E507K and E742K; the nucleotide molecular sequence encoding the above mutant Taq DNA polymerase is as shown in SEQ ID NO:3.

[0041] The present invention uses directed evolution technology to screen out amino acid sites and their mutation methods that are highly correlated with the activity of Taq DNA polymerase from a random mutation library. Specifically, in each round, it is possible to screen out the target sequence from 8×10 7 mutants. Compared with site-directed mutagenesis, the number of mutants obtained by this technology is 10 5 times that of site-directed mutagenesis, which is conducive to screening out mutant sites with synergistic effects, and these mutant sites cannot be predicted by existing computer simulation technologies. At the same time, based on the principle of directed evolution, the accumulated advantageous traits are the most adaptable to the added screening conditions, and the corresponding mutants obtained are also the individuals with the optimal target performance among all mutants.

[0042] The mutant Taq DNA polymerase provided by the present invention can be expressed or produced through the following steps:

[0043] (1) Transform or transduce a suitable host cell with the polynucleotide sequence encoding the protein of the present invention as shown in SEQ ID NO:3, or the polynucleotide sequence reverse-derived from the amino acid sequence SEQ ID NO:4, or a recombinant expression vector containing the polynucleotide;

[0044] (2) Culture and induce the host cell to express the target protein in a suitable medium;

[0045] (3) Isolate and purify the target protein from the culture medium or cells to obtain the fast Taq DNA polymerase, i.e., the mutant Taq DNA polymerase.

[0046] Construct an expression vector containing the coding nucleotide sequence of the mutant Taq DNA polymerase of the present invention and appropriate transcriptional and translational control signals. Preferably, the vector is: pET-28a. After the nucleotide sequence is ligated to an appropriate promoter in the expression vector by DNA ligase, it directs the synthesis of mRNA. The expression vector also includes elements such as ribosome binding sites, operons, fusion tags, transcriptional terminators, and selectable marker genes.

[0047] The expression vector of the present invention can be used to transform appropriate host cells so that the host transcribes the target RNA or expresses the target protein. The host cells can be prokaryotic cells, such as Escherichia coli, Bacillus subtilis, Streptomyces; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as animal cells. Deliver the recombinant DNA to the host cells. For example, when the host is Escherichia coli, transformation techniques such as the CaCl2 method and electroporation can be used; when the host is a eukaryotic animal cell, DNA transfection techniques such as calcium phosphate co-precipitation, microinjection, electroporation, and liposome packaging can be selected; when the host is a eukaryotic plant cell, techniques such as Agrobacterium transformation or gene gun transformation can be used.

[0048] After obtaining the engineered cells, culture the engineered cells under suitable conditions to express the target protein encoded by the polynucleotide sequence of the present invention. In the present invention, conventional fermentation conditions can be used, such as fermenting using LB, TB, and self-inducing media at a temperature of 25-37°C.

[0049] The target protein Taq DNA polymerase of the present invention exists intracellularly in Escherichia coli cells. Use a centrifuge to collect the host cells, and then break the host cells by high-pressure homogenization, ultrasound, enzymatic cell lysis, or other cell disruption methods to release the recombinant protein. Preferably, it is the high-pressure homogenization method. The lysed bacterial solution is initially purified by heat treatment. For example, heat treat at 70°C for 30 min, invert and mix evenly every 10 min, and then centrifuge to remove heat-labile denatured impurity proteins. The resulting feed solution is then subjected to purification processes such as chromatography and ultrafiltration to obtain high-purity mutant Taq DNA polymerase. Specific chromatography techniques include: affinity chromatography, ion exchange, hydrophobic chromatography, and molecular sieve techniques; ultrafiltration media include: polysulfone membranes, ceramic membranes, metal membranes, etc.

[0050] The present invention also provides a fast PCR amplification kit, which includes: 5×Fast buffer and mutant Taq DNA polymerase; wherein, the 5×Fast buffer includes a buffer pair, a monovalent cation, an enhancer, and a surfactant. Specifically:

[0051] The amount of mutant Taq DNA polymerase ≥ 5 U / 25 μL; preferably, the amount of mutant Taq DNA polymerase is 5 U - 10 U / 25 μL.

[0052] The buffer pair uses Tris-HCl, and the pH value of Tris-HCl can be arbitrarily selected within the range of 8.4 - 9.6, such as any one or the range between any two of 8.4, 8.6, 8.8, 9.0, 9.2, 9.4, and 9.6.

[0053] In a preferred embodiment, the final concentration of Tris-HCl in 5×Fast buffer can be arbitrarily selected within the range of 50 - 250 mmol / L, such as arbitrarily selected within the range of 62.75 - 250 mmol / L, such as any one or the range between any two of 50 mmol / L, 70 mmol / L, 90 mmol / L, 110 mmol / L, 130 mmol / L, 150 mmol / L, 170 mmol / L, 190 mmol / L, 210 mmol / L, 230 mmol / L, and 250 mmol / L. Within this range, effective amplification can be achieved.

[0054] The monovalent cation includes NH4 + When it is NH4 + The concentration can be arbitrarily selected within the range of 100 - 300 mmol / L, such as any one or the range between any two of 100 mmol / L, 125 mmol / L, 150 mmol / L, 175 mmol / L, 200 mmol / L, 45 mmol / L, 250 mmol / L, 275 mmol / L, and 300 mmol / L. NH4 + The ionization of NH4 ions to hydrogen ions has reversible competition, which can improve the binding force between the specifically bound primer and template, provide hydrogen ions, and reduce the non-specific binding force by competitively capturing hydrogen ions, improve the stringency of annealing conditions, further reduce non-specific amplification during the reaction, and significantly improve the specificity and sensitivity of the PCR reaction. Preferably, the ammonium salt is selected from at least one of (NH4)2SO4, NH4Cl, and CH3COONH4, and more preferably (NH4)2SO4.

[0055] The monovalent cation includes K + When it is K, the final concentration of the potassium salt can be arbitrarily selected within the range of 50 - 300 mmol / L, such as any one or the range between any two of 50 mmol / L, 100 mmol / L, 150 mmol / L, 200 mmol / L, 250 mmol / L, and 300 mmol / L. K +Combining with the phosphate groups on the template and primers to neutralize the excessive negative charge of DNA can improve the template-primers binding force and further enhance the amplification efficiency. Preferably, K + is selected from any one of KCl and K2SO4, and more preferably KCl.

[0056] Enhancer, the final concentration of the enhancer is arbitrarily selected within the range of 0.02% - 0.1 (v / v)%, specifically any value or the range between any two values among 0.02 (v / v)%, 0.04 (v / v)%, 0.06 (v / v)%, 0.08 (v / v)%, and 0.1 (v / v)%. Preferably, the enhancer is selected from at least one of BSA and acetylated BSA, and more preferably BSA.

[0057] In a preferred embodiment, the enhancer further includes TMAC, and its final concentration in the 5×Fast buffer is arbitrarily selected within the range of 150 - 500 mmol / L, specifically any value or the range between any two values among 150 mmol / L, 200 mmol / L, 250 mmol / L, 300 mmol / L, 350 mmol / L, 400 mmol / L, 450 mmol / L, and 500 mmol / L. TMAC improves the specificity of hybridization, that is, reduces non-specific binding, thereby enhancing the accuracy and efficiency of the PCR reaction. TMAC can also increase the melting temperature of DNA, which means that during the PCR cycle, DNA can still maintain a double-stranded structure at a higher temperature, facilitating subsequent amplification reactions. This effect helps to eliminate non-specific priming, reduce the misbinding of DNA and RNA, and thus ensure the purity and accuracy of the PCR products.

[0058] Surfactant, the volume fraction of the surfactant is arbitrarily selected within the range of 0.1 - 1 (v / v)%, such as any value or the range between any two values among 0.1 (v / v)%, 0.2 (v / v)%, 0.3 (v / v)%, 0.4 (v / v)%, 0.5 (v / v)%, 0.6 (v / v)%, 0.7 (v / v)%, 0.8 (v / v)%, 0.9 (v / v)%, and 1 (v / v)%. Preferably, the surfactant is selected from any one of Tween 20, CA-630, and Triton X-100, and more preferably Triton X-100.

[0059] Furthermore, the kit further includes at least one of dNTP mixture, primer pair, Mg 2+ and water.

[0060] The present invention also provides a method for rapid PCR amplification, which includes performing PCR amplification by using the hot-start fast Taq DNA polymerase in the kit and the PCR buffer 5×Fast buffer.

[0061] Mix the DNA template, primer pair, Taq Man probe, 5×Fast buffer, dNTPs, Mg 2+ and the hot-start fast Taq DNA polymerase, and then perform amplification based on the PCR reaction conditions.

[0062] Mg 2+ is one of the essential components for the activity of Taq DNA polymerase. In addition to affecting the enzyme activity and fidelity, the Mg 2+ concentration also affects primer annealing, the melting temperature of the template and PCR products, the specificity of the products, and the formation of primer dimers, etc. When the Mg 2+ concentration is too low, the enzyme activity is significantly reduced; when it is too high, the enzyme can catalyze non-specific amplification. In a preferred embodiment, the Mg 2+ concentration can be arbitrarily selected within the range of 2-8 mmol / L, for example, any one or the range between any two of 2 mmol / L, 3 mmol / L, 4 mmol / L, 5 mmol / L, 6 mmol / L, 7 mmol / L, and 8 mmol / L.

[0063] For rapid PCR amplification, the total amplification reaction time is less than 1 h. In some embodiments, the total amplification reaction time is less than 50 min, less than 40 min, less than 30 min, less than 20 min, or less than 10 min.

[0064] In a preferred embodiment, the reaction program for rapid PCR amplification is selected from any one of Program A and Program B:

[0065] Program A includes: 93-98°C, 30-90 sec; (95-99°C, 0.5-3 sec; 45-65°C, 4-6 sec; 70-74°C, 4-6 sec)×40;

[0066] Program B includes: 93-98°C, 30-90 sec; (95-99°C, 0.5-3 sec; 58-62°C, 5-10 sec)×40;

[0067] Based on the Gentier 48E fully automatic PCR analysis system, in the module temperature control mode, the present invention adopts the program: 98°C, 30 sec; (97°C, 3 sec; 60°C, 5 sec)×40 Cycle for two-step amplification, and can achieve 40 cycles of amplification in 19 min, with a net time of only 350 sec, significantly reducing the time required for the PCR amplification reaction.

[0068] It is understandable that the components and their concentrations of the rapid PCR buffer in this embodiment can be the same as those described in any of the foregoing corresponding embodiments or implementation manners, and will not be elaborated herein.

[0069] Example 1

[0070] The CSR method was used to screen for mutant Taq DNA polymerase, specifically as follows:

[0071] 1. Establishment of a random mutation library

[0072] The mutation library is the first and most crucial step in directed evolution. The method for constructing a random mutation library is as follows:

[0073] Using the circular plasmid containing pET-28a-Taq DNA polymerase, the wild-type Taq DNA polymerase is derived from Thermus aquaticus, and the nucleotide sequence of the wild-type Taq DNA polymerase is shown in SEQ ID NO:1 as a template for PCR amplification. The random mutation amplification system is shown in Table 1. The 2×PCR buffer formulation is: 20 mmol / L Tris-HCl (pH 9.0, 25 °C), 100 mmol / L KCl, 5 mmol / L MgCl2, 0.2 (v / v)% Triton X-100; primer F1: 5'-GAAGGAGATATACCATGGGCAGC-3', primer R1: 5'-TGCTAGTTATTGCTCAGCGGTGG-3'. The random mutation amplification program shown in Table 2 was used for amplification. After amplification, the amplification product was digested with Takara Dpn I to remove the original plasmid template, and then gel extraction was performed for standby.

[0074] Table 1: Random mutation PCR system

[0075] System Component Name / Specification Dosage (μL) 2×PCR buffer 25 Primer F1 (10 μM) 1 Primer R1 (10 μM) 1 dATP (10 mmol / L) 1 dGTP (10 mmol / L) 1 dCTP (10 mmol / L) 5 dTTP (10 mmol / L) 5 Hot-Start Taq DNA Polymerase (5 U / μL) 1 <![CDATA[MnCl2(10 mmol / L)]]> 0.5 / 1 / 1.5 / 2 / 2.5 Template (pET-28a-Taq plasmid) (10 ng / μL) 2 <![CDATA[ddH2O]]> Up to 50

[0076] Note: The mutation rate can be controlled by the addition amount of MnCl2. The higher the addition amount of MnCl2, the higher the mutation rate. The mutation rate of 10 mmol / L MnCl2 under the addition amount condition of 1 μL / T is approximately 0.33%.

[0077] Table 2: Random mutation amplification program

[0078]

[0079] The PCR products recovered by cutting the gel were digested with the restriction enzymes Nde I and Xhol I, and then ligated to the pET-28a vector linearized with the restriction enzymes Nde I and Xhol I under the action of T4 DNA Ligase. Take 10 μL of the reaction system and add it to 50 μL of BL21(DE3) Chemically Competent Cell, which is the expression host cell. Incubate on ice for 30 min, heat shock at 42 °C for 90 sec, place on ice for 2 min, add 950 μL of LB medium, and incubate in a shaker at 37 °C with a rotation speed of 200 rpm for 1 h.

[0080] The random mutant library of Taq DNA polymerase was constructed by the above method and introduced into BL21(DE3) Chemically Competent Cell for CSR screening.

[0081] 2. Directed evolution under rapid amplification pressure

[0082] Centrifuge the activated transformation solution at 2000 rpm and 4 °C for 3 min, remove 900 μL of the supernatant, resuspend the remaining part, and inoculate all of it into 10 mL of LB liquid medium containing 50 μg / mL kanamycin. Culture overnight at 37 °C and 200 rmp for 12 - 14 h. Take 2 mL of the overnight culture and inoculate it into 200 mL of LB liquid medium containing 50 μg / mL kanamycin. Culture at 37 °C and 200 rmp until OD 600 = 0.6 - 0.9, add IPTG inducer with a final concentration of 0.5 mmol / L, and continue shaking the bacteria at 37 °C and 200 rpm for 2 - 4 h. Centrifuge at 3000 rpm for 15 min to collect the bacteria, resuspend and wash the bacteria with 1×PCR buffer with a volume of 1 / 10 of the medium volume. Repeat centrifuging at 3000 rpm for 15 min to collect the bacteria, and then resuspend the bacteria with an appropriate amount of 1×PCR buffer to make the OD of the resuspension 600 = 0.5 - 1. The formula of 1×PCR buffer is: 10 mmol / L Tris-HCl, pH 9.0, 25 °C, 50 mmol / L KCl, 2.5 mmol / L MgCl2, 0.1 (v / v)% Triton X-100.

[0083] Prepare the CSR oil phase: 4.5 (v / v)% span 80, 4.0 (v / v)% Tween-80, 0.05 (v / v)% Triton X-100, 91.45 (v / v)% light mineral oil, as shown in Table 3.

[0084] Table 3: Preparation method of CSR oil phase

[0085] Component Volume span 80 2250 μL Tween-80 2000 μL TritonX-100 25 μL Light Mineral Oil 45.75 mL

[0086] Prepare the CSR aqueous phase: 1 μmol / L primer, 0.25 mmol / L dNTP, and make up the total volume with a bacterial suspension with OD600 = 0.5 - 1, as shown in Table 4.

[0087] Table 4: Preparation method of CSR aqueous phase

[0088] Component / Specification Volume <![CDATA[Bacterial suspension (OD 600 = 0.5 - 1)]]> 191 μL Primer F1 (100 μM) 2 μL Primer R1 (100 μM) 2 μL dNTP (10 mmol / L) 5 μL

[0089] Emulsification: Place a rotor in a 2 mL flat-bottomed tube, add 400 μL of CSR oil phase, adjust the rotation speed to 1800 rpm and stir. Dropwise add 200 μL of CSR aqueous phase into 400 μL of CSR oil phase, 5 - 10 μL per drop, with an interval of 5 s. After adding the last drop, continue to stir for 30 min. After emulsification, it is in a white and viscous state. Aliquot the emulsion at 50 μL / T to the bottom of a PCR tube, add a drop of light mineral oil on the top layer to form a liquid seal, and perform amplification using the program shown in Table 5. The full length of Taq DNA polymerase is 2496 bp, and set an extension time of 5 s for pressure screening. The state after emulsion PCR amplification should be non-layered, as Figure 1 shown; if it is layered, it indicates that the emulsification process is incomplete and should be discarded.

[0090] Table 5: Emulsion PCR amplification program

[0091]

[0092] Extraction of emulsion PCR products: After the emulsion PCR is completed, recover all the emulsion PCR products and combine them in a centrifuge tube. After centrifugation at 12000 rpm for 10 min, discard the upper oily substance. In the remaining solution, vortex mix with 6 - 10 times of Binding solution of Tiangen DNA purification kit, then add it to the adsorption column. After centrifugation at 12000 rpm for 1 min, wash twice with Washingsolution at 12000 rpm for 30 s and centrifuge the empty column once. Add ddH2O and centrifuge at 12000 rpm for 1 min to complete the recovery. However, due to the severity of the screening pressure, there may be cases where the PCR products cannot be recovered in multiple experiments in this step.

[0093] Digest the original template with Takara Dpn I for the successfully recovered amplified products, then amplify the recovered products using TOYOBO high-fidelity KOD DNA polymerase. The reaction system and conditions refer to the instruction manual, and perform gel extraction and recovery.

[0094] The recovered product was digested with restriction endonucleases Nde I and Xhol I, connected to the vector and transformed into BL21 (DE3) Chemically Competent Cell competent cells, and aseptically coated on LB solid plates containing 50 μg / mL kanamycin.

[0095] 3. Second pressure screening of mutants

[0096] Aseptically pick the monoclonal colony from the LB solid plate into a 96-well plate containing 2 mL of autoinduction medium, and culture at 37°C and 200 rpm for 12-16 h. Take 2 μL of the fermentation product directly into the system shown in Table 6 and amplify using the program shown in Table 7. The amplicon is a 2500 bp DNA fragment, primer F2: 5'-CATCCGACATCCACAGGTC-3', primer R2-4: 5'-ACCCTTACCCCAGAAGTCAT-3'. A total of 200 single colonies were picked, and only one single colony amplified the target band, while the rest had no target band, such as Figure 2 As shown, colony 52 has a fragment of the amplified target length. The bacterial stock solution was sent to a third-party sequencing company for sequencing. The sequencing result is shown in SEQ ID NO: 3. The Clustal Omega sequence alignment tool was used to align the sequencing result sequence with the wild-type sequence, as shown in Figure 3 As shown, a total of 8 mutations occurred in the sequencing results. Due to the degeneracy of the codons, 5 mutations occurred in the amino acid sequence, namely D58E, K292N, G304A, E507K, and E742K. Thus, the present invention successfully screened a mutant Taq DNA polymerase with rapid amplification ability, namely, strain Mut 52.

[0097] Table 6: Second pressure screening PCR system

[0098] System Component Name / Specification Volume (μL) 2×PCR buffer 12.5 <![CDATA[MgCl2(50 mmol / L)]]> 1 Primer F1 (10 μM) 0.5 Primer R4 (10 μM) 0.5 dNTP (10 mmol / L) 0.5 Bacterial Solution (Providing Fast Taq DNA Polymerase) 2 pPIC3.5K Plasmid Template (10 ng / μL) 1 <![CDATA[ddH2O]]> Up to 25

[0099] Table 7: Second pressure screening PCR program

[0100]

[0101] Example 2

[0102] The purification and preparation of mutant Taq DNA polymerase are as follows:

[0103] 1. Fermentation

[0104] Pick the monoclonal colonies identified by sequencing in Example 1, and inoculate them aseptically into 10 mL of LB medium containing 50 μg / mL kanamycin resistance. Incubate with shaking at 37°C and 200 rpm overnight. The next day, transfer all of them to 1000 mL of auto-induction medium containing 50 μg / mL kanamycin resistance, and incubate with shaking at 37°C and 200 rpm for 12 - 16 h. Centrifuge at 9000 rpm for 5 min to collect the bacterial cells for later use.

[0105] 2. Purification

[0106] Weigh the bacterial cells collected by fermentation and use Lysis buffer. Use 10 mL of Lysis buffer for 1 g of cells. After fully resuspending the bacterial cells with a glass rod, add PMSF with a volume of one-thousandth of the Lysis buffer. Then homogenize 3 times under the condition of 850 bar using a high-pressure homogenizer. Aliquot the lysed bacterial solution into centrifuge buckets, balance them, and place them in a pre-cooled low-temperature centrifuge. Centrifuge at 9000 rpm and 4°C for 20 min to collect the supernatant after centrifugation. Incubate the centrifuged supernatant in a water bath at 70°C for 30 min, and invert and mix it every 10 min during this period. Aliquot the heat-treated bacterial solution into centrifuge buckets, balance them, and place them in a pre-cooled low-temperature centrifuge. Centrifuge at 9000 rpm and 4°C for 20 min to collect the supernatant after centrifugation. Use a protein purification system to purify the collected supernatant through one affinity chromatography and one anion exchange chromatography to obtain mutant Taq DNA polymerase with a purity of over 99%. The SDS-PAGE electrophoresis result after purification is as Figure 4 shown.

[0107] After measuring the enzyme activity of the high-purity mutant Taq DNA polymerase according to the enzyme activity measurement method in the national standard GB / T 35542 - 2017 "Taq DNA Polymerase", dilute it to the working concentration and add an appropriate amount of antibody to prepare a 5 U / μL hot-start rapid Taq DNA polymerase.

[0108] Example 3

[0109] Test on the extension rate of mutant Taq DNA polymerase is as follows:

[0110] To detect the amplification rate of the mutant Taq DNA polymerase, in this invention, the wild-type Taq DNA polymerase was used as a control group. Using the pPIC3.5K plasmid vector as a template, a common upstream primer F2 and multiple downstream primers: R2-1, R2-2, R2-3, R2-4, R2-5, and R2-6 were designed to amplify amplicons of 700bp, 1500bp, 2000bp, 2500bp, 3000bp, and 3500bp respectively, as shown in Table 8. The PCR system shown in Table 9 and the program shown in Table 10 were used for amplification. After the reaction ended, the amplification results were detected by 1.0 (m / v)% agarose gel electrophoresis, and the amplification rate was calculated.

[0111] The results of agarose electrophoresis showed that, as Figure 5 shown, in this system, the Mut 52 mutant could complete the amplification of the 3000bp amplicon, and the product amount was basically the same as that of the smaller molecular weight amplicons, and the band brightness was also basically the same. It was calculated that the amplification rate of the Mut 52 mutant could reach 600bp / sec, while the wild-type Taq DNA polymerase could only fully amplify the 750bp molecular weight fragment, and the product amount of the higher molecular weight amplification was significantly reduced. It was calculated that the amplification rate of the wild-type Taq DNA polymerase was about 150bp / sec. Thus, it can be seen that the amplification rate of the Mut 52 mutant is 4 times that of the wild-type Taq DNA polymerase.

[0112] Table 8: Primer sequences for detecting the amplification rate

[0113] Primer Name Primer Sequence Amplicon Length Primer F2 5’-CATCCGACATCCACAGGTC-3’ / Primer R2-1 5’-AAGACAGGGCAGCTTCCTTC-3’ 750 bp Primer R2-2 5’-GAGAACGGGTGCGCATAGAAA-3’ 1500 bp Primer R2-3 5’-TGTCCAATCAGTACGCTCCT-3’ 2000 bp Primer R2-4 5’-ACCCTTACCCCAGAAGTCAT-3’ 2500 bp Primer R2-5 5’-CTGAAGGTCGTATTGAATTGTGC-3’ 3000 bp Primer R2-6 5’-GAGTCTACCAATCTCGTCGTCAC-3’ 3500 bp

[0114] Table 9: PCR system for detecting the amplification rate

[0115]

[0116]

[0117] Table 10: PCR program for detecting the amplification rate

[0118]

[0119] Example 4

[0120] Test for the anti-inhibition property of the mutant Taq DNA polymerase is as follows:

[0121] To detect the anti-inhibition property of the mutant Taq DNA polymerase, the present invention uses the wild-type Taq DNA polymerase as a control group, prepares the PCR system as shown in Table 11, and performs qPCR amplification using the procedure shown in Table 12, with two replicates for each sample. The inhibitors include whole blood, plasma, serum, SDS, porcine bile salt, ethanol, and M-MLV reverse transcriptase.

[0122] The test results are shown in Table 13. The wild-type Taq enzyme is not resistant to whole blood, while the mutant Taq DNA polymerase can tolerate 1.25 (v / v)% whole blood, and only delays by 0.394 Ct under the condition of 2.5 (v / v)% whole blood, showing good resistance to whole blood; the wild-type Taq enzyme is not resistant to plasma, while the mutant Taq DNA polymerase can tolerate 10 (v / v)% plasma; the wild-type Taq enzyme is not resistant to serum, while the mutant Taq DNA polymerase can tolerate 10 (v / v)% serum; the wild-type Taq enzyme can tolerate 0.0125 (m / v)% SDS, while the mutant Taq DNA polymerase can tolerate 0.025 (m / v)% SDS; the test results of resistance to porcine bile salt show that the performance of the wild-type Taq enzyme and the mutant Taq DNA polymerase is basically the same, and both can tolerate 0.02 (m / v)% porcine bile salt; the wild-type Taq enzyme can tolerate 2 (v / v)% ethanol, while the mutant Taq DNA polymerase can tolerate 4 (v / v)% ethanol; the wild-type Taq enzyme can tolerate 20 U / T of M-MLV reverse transcriptase, while the mutant Taq DNA polymerase can tolerate 200 U / T of M-MLV reverse transcriptase. Thus, it can be seen that the mutant Taq DNA polymerase provided by the present invention has excellent inhibitor resistance.

[0123] Table 11: PCR system for anti-inhibition test

[0124] System Component Name Volume (μL) 2×PCR buffer 12.5 <![CDATA[MgCl2(50 mmol / L)]]> 1 dNTP (10 mmol / L) 0.5 Primer Probe Mix 1 Wild-Type Taq DNA Polymerase / Mut 52DNA Polymerase 0.5 Inhibitor 2 Template 5 <![CDATA[ddH2O]]> Up to 25

[0125] Table 12: PCR procedure for anti-inhibition test

[0126]

[0127]

[0128] Table 13: Test results of anti-inhibition

[0129]

[0130] Note: "-" indicates that the PCR instrument did not collect fluorescence signals; "#DIV / 0!" indicates a calculation error due to incomplete data.

[0131] Example 5

[0132] The heat resistance test of the mutant Taq DNA polymerase is as follows:

[0133] To detect the heat resistance of the mutant Taq DNA polymerase, the wild-type Taq DNA polymerase was used as a control group in this invention. Both enzymes were heat-treated at 95 °C for 1 h and 2 h. After heat treatment, qPCR amplification was performed using the PCR system shown in Table 14 and the program shown in Table 12, with two replicates for each sample. The test results of the wild-type Taq DNA polymerase are as Figure 6 shown in A. After heat treatment, the Ct value did not change significantly, but the fluorescence end point value decreased significantly after treatment at 95 °C for 1 h and 2 h. For the mutant Taq enzyme, as Figure 6 shown in B, after heat treatment at 95 °C for 1 h and 2 h, the Ct value did not change significantly, and the fluorescence end point value was also consistent with the control, indicating that the thermal stability of the mutant Taq DNA polymerase is significantly higher than that of the wild-type Taq enzyme.

[0134] Table 14: PCR system for heat resistance test

[0135] System Component Name Volume (μL) 2×PCR buffer 12.5 <![CDATA[MgCl2(50mmol / L)]]> 1 dNTP (10 mmol / L) 0.5 Primer Probe Mix 1 Wild-Type Taq DNA Polymerase / Mut 52DNA Polymerase 0.5 Template 5 <![CDATA[ddH2O]]> Up to 25

[0136] Example 6

[0137] Debugging of the fast PCR buffer is as follows:

[0138] This example provides a 5×Fast buffer for fast PCR, and its components are shown in Table 15:

[0139] Table 15: Components of 5×Fast buffer

[0140] Component Final Concentration Tris-HCl pH 9.4 150 mmol / L KCl 250 mmol / L Triton X-100 0.5 (v / v)% BSA 0.1 (v / v)% TMAC 300 mmol / L

[0141] This example also provides a method for fast PCR amplification. Using the above 5×Fast buffer and the mutant Taq DNA polymerase Mut 52 at 5 U / μL for fast PCR amplification, the specific steps are as follows:

[0142] Prepare the corresponding reaction system using 5×Fast buffer and mutant Taq DNA polymerase.

[0143] The fast PCR reaction conditions are as follows: Based on the full-automatic PCR analysis system, in the module temperature control mode, use the program: 98 °C, 30 sec; (97 °C, 3 sec; 60 °C, 5 sec) × 40 Cycle for two-step amplification, achieving 40 cycles of amplification in 19 min, with a net time of only 350 sec.

[0144] Verification Example 1: Screening of buffer systems

[0145] Multiple sets of rapid PCR buffers were set up. Compared with Table 15, the difference lies in the different pH values and the molar amounts of buffer pairs in the buffers. By the Taq Man probe method, the PCR system in Table 16 was used to verify the effect of different buffer pH values on the amplification effect. The amplification results are the average Ct values of two replicates. Program 1: 95°C, 90 sec; (97°C, 5 sec; 60°C, 10 sec) × 40 cycles. The detection results of buffers with different pH values are shown in Table 17. At pH 9.4, the amplification Ct values of the two templates are relatively the smallest, which is the optimal amplification result.

[0146] Table 16: PCR System

[0147]

[0148]

[0149] Table 17: Detection Ct Value Results of Buffers with Different pH Values

[0150] pH Gradient pH 8.4 pH 8.6 pH 8.8 pH 9.0 pH 9.2 pH 9.4 pH 9.6 Template 1 32.465 31.835 31.667 31.481 31.128 30.539 31.225 Template 2 35.751 35.146 34.946 34.872 34.411 33.927 34.327

[0151] Furthermore, based on the optimal pH value, the PCR system in Table 16 was used to verify the effect of different molar amounts of buffer pairs on the amplification effect. The amplification results are the average Ct values of two replicates. Program 1: 95°C, 90 sec; (97°C, 5 sec; 60°C, 10 sec) × 40 cycles. The detection Ct value results of different molar amounts of buffer pairs are shown in Table 18. When the molar amount of the buffer pair is 150 mmol / L, the amplification Ct values of the two templates are the smallest, which is the optimal amplification result. Therefore, the buffer system is selected as 150 mmol / L Tris-HCl pH 9.4 @ 25°C.

[0152] Table 18: Detection Ct Value Results of Different Molar Amounts of Buffer Pairs

[0153] Buffer Pair Molarity 50 mmol / L 100 mmol / L 150 mmol / L 250 mmol / L Template 1 30.563 30.489 30.196 30.231 Template 2 34.652 34.473 34.004 34.500

[0154] Verification Example 2: Screening of Monovalent Cations

[0155] Based on the optimal conditions screened in Verification Example 1, multiple sets of rapid PCR buffers were set up. Compared with Table 15, the difference is that the monovalent cations are K + and NH4 +, and different concentrations were set. By the Taq Man probe method, the PCR system in Table 16 was used to verify the effects of different monovalent cations and their different concentrations on the amplification effect. The amplification results were the average Ct values of two replicates. Program 1: 95°C, 90 sec; (97°C, 5 sec; 60°C, 10 sec) × 40 Cycle. The results of the Ct values detected for the types and concentrations of monovalent cations are shown in Table 19. The results showed that when using 250 mmol / L K + The Ct value of the amplification was the smallest, which was the optimal amplification result. Therefore, 250 mmol / L potassium chloride was selected as the monovalent cation.

[0156] Table 19: Results of Ct values detected for the types and concentrations of monovalent cations

[0157]

[0158] Verification Example 3: Reduction of the time for each step of the amplification program

[0159] Based on the optimal conditions screened in Verification Example 2, multiple groups of rapid PCR buffers were set. On the basis of Table 15, the concentration of the enhancer and the amount of mutant Taq DNA polymerase were adjusted respectively.

[0160] Reduction of the extension time: The enhancers TMAC and the super-thermostable single-stranded binding protein ET SSB that may achieve a reduction in the extension time were added to the rapid PCR buffer respectively, and the amount of mutant Taq DNA polymerase was adjusted. By the Taq Man probe method, the PCR system in Table 16 was used to verify the effects of different enhancers and the amount of mutant Taq DNA polymerase on the amplification effect. The amplification results were the average Ct values of two replicates,

[0161] Program 1: 95°C, 90 sec; (97°C, 5 sec; 60°C, 10 sec) × 40 Cycle;

[0162] Program 2: 95°C, 90 sec; (97°C, 5 sec; 60°C, 5 sec) × 40 Cycle.

[0163] The amplification results showed that, as shown in Table 20, after amplification by Program 2, after adding 300 mmol / L TMAC to the 5×Fast buffer, △Ct approached 0, the Ct value was significantly advanced, and it was close to the Ct value of the control group Program 1; while for the groups adding different amounts of ETSSB, the Ct values were all significantly delayed, and this component had a significant inhibitory effect on the system; therefore, the optimal addition concentration of TMAC was determined to be 300 mmol / L. On this basis, the amount of hot-start rapid Taq DNA polymerase was increased to 1.6 μL / T, and the Ct value of Program 2 was basically the same as that of Program 1, achieving the reduction of the extension time from 10 sec to 5 sec.

[0164] Table 20: Results of Ct values detected by adjusting the amounts of TMAC, ET SSB and Taq DNA polymerase

[0165]

[0166] Note: "-" indicates that the PCR instrument did not collect fluorescence signals; "#DIV / 0!" indicates a calculation error caused by incomplete data.

[0167] Reducing the denaturation time: Tests were set up to directly shorten the denaturation time without adding additives, and to add formamide and tetrahydropyrimidine, which may reduce the denaturation time, to the PCR reaction system. The effects of different additives on the denaturation effect were verified by the Taq Man probe method using the PCR system in Table 16. The amplification results were the average Ct values of two replicates.

[0168] Program 2: 95°C, 90 sec; (97°C, 5 sec; 60°C, 5 sec) × 40 cycles

[0169] Program 3: 95°C, 90 sec; (97°C, 3 sec; 60°C, 5 sec) × 40 cycles

[0170] Program 4: 95°C, 90 sec; (97°C, 1 sec; 60°C, 5 sec) × 40 cycles

[0171] The amplification results showed that, as shown in Table 21, when using Program 3 to shorten the denaturation time to 3 sec for detection, the Ct value results were basically the same as the control, with △Ct ≤ 0.2; however, when using Program 4 for amplification, the Ct was delayed by more than 3. As shown in Table 22, under the conditions of Program 4, adding tetrahydropyrimidine and formamide with strand separation functions did not have obvious effects. Thus, it can be seen that the mutant Taq DNA polymerase provided in this application can complete amplification under the denaturation conditions of Program 3.

[0172] Table 21: Results of Ct values detected by directly shortening the denaturation time

[0173]

[0174] Note: "-" indicates that the PCR instrument did not collect fluorescence signals; "#DIV / 0!" indicates a calculation error caused by incomplete data.

[0175] Table 22: Results of Ct values detected by adding additives on the basis of shortening the denaturation time

[0176]

[0177] Note: "-" indicates that the PCR instrument did not collect fluorescence signals; "#DIV / 0!" indicates a calculation error caused by incomplete data.

[0178] Reducing the deblocking time: Based on the system with shortened denaturation and extension times, the present invention adjusts the deblocking time. By the Taq Man probe method, the PCR system in Table 16 was used to verify the effect of deblocking at different times on the amplification effect. The amplification results are the average Ct values of two replicates.

[0179] Program III: 95°C, 90 sec; (97°C, 3 sec; 60°C, 5 sec) × 40 Cycles;

[0180] Program V: 95°C, 60 sec; (97°C, 3 sec; 60°C, 5 sec) × 40 Cycles;

[0181] Program VI: 95°C, 30 sec; (97°C, 1 sec; 60°C, 5 sec) × 40 Cycles;

[0182] Program VII: 98°C, 30 sec; (97°C, 3 sec; 60°C, 5 sec) × 40 Cycles;

[0183] Program VIII: 98°C, 15 sec; (97°C, 3 sec; 60°C, 5 sec) × 40 Cycles.

[0184] The amplification results showed that, as shown in Table 23, when using Program VI and Program VIII for amplification, there was a delay of about 0.3 CT, not reaching the target; when using Program V and Program VII, the amplification results were consistent with the control, ΔCt ≤ 0.2. Therefore, Program VII was the optimal result with the shortest time, achieving 40 cycles of amplification in 19 min, and the net time was only 350 sec.

[0185] Table 23: Results of detecting Ct values for shortening the deblocking time

[0186]

[0187] Example 7

[0188] Verification of amplification performance is as follows:

[0189] Prepare the system shown in Table 16, and use the mutant Taq DNA polymerase prepared in Example 2 and the 5×Fast buffer adjusted in Example 4 to perform amplification tests on the high-concentration template and the linearly gradient template diluted by 10 n to detect the performance of the rapid detection system. At the same time, test the amplification performance of the 5×Fast buffer after being repeatedly frozen and thawed 20 times. The test results are shown in Table 24 and Figure 7A, 7B. The amplification results using the rapid detection program seven are basically the same as the Ct values of the non-rapid detection program one, with △Ct ≤ 0.2, and the linear pattern is basically the same as the control, indicating that this mutant Taq DNA polymerase can detect high, medium, and low concentration targets when matched with 5×Fast buffer; as shown in Table 24 and Figure 7 A, 7C. The amplification results using the 5×Fast buffer that has been frozen and thawed 20 times with the rapid detection program seven are basically the same as the Ct values of the non-rapid detection program one, with △Ct ≤ 0.2, and the linear pattern is basically the same as the control, indicating that the performance of the 5×Fast buffer is stable after 20 repeated freeze-thaw tests.

[0190] Table 24: Results of Ct values for linear gradient template detection

[0191]

[0192]

[0193] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

[0194] The sequences mentioned in this article are specifically as follows:

[0195] SEQ ID NO:1 (The nucleotide sequence of wild-type Taq DNA polymerase is as follows):

[0196]

[0197] SEQ ID NO:2 (The amino acid sequence of wild-type Taq DNA polymerase is as follows):

[0198] MRGMLPLFEPKGRVLLVDGHHLAYRTFHALKGLTTSRGEPVQAVYGFAKSLLKALKEDGDAVIVVFDAKAPSFRHEAYGGYKAGRAPTPEDFPRQLALIKELVDLLGLARLEVPGYEADDVLASLAKKAEKEGYEVRILTADKDLYQLLSDRIHVLHPEGYLITPAWLWEKYGLRPDQWADYRALTGDESDNLPGVKGIGEKTARKLLEEWGSLEALLKNLDRLKPAIREKILAHMDDLKLSWDLAKVRTDLPLEVDFAKRREPDRERLRAFLERLEFGSLLHEFGLLESPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEEIARLEAEVFRLAGHPFNLNSRDQLERVLFDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDLIHPRTGRLHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQIELRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINFGVLYGMSAHRLSQELAIPYEEAQAFIERYFQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWLSAKE

[0199] SEQ ID NO:3 (The nucleotide sequence of mutant Taq DNA polymerase is as follows):

[0200]

[0201] SEQ ID NO: 4 (Amino acid sequence of the mutant Taq DNA polymerase is as follows):

[0202] MRGMLPLFEPKGRVLLVDGHHLAYRTFHALKGLTTSRGEPVQAVYGFAKSLLKALKEEGDAVIVVFDAKAPSFRHEAYGGYKAGRAPTPEDFPRQLALIKELVDLLGLARLEVPGYEADDVLASLAKKAEKEGYEVRILTADKDLYQLLSDRIHVLHPEGYLITPAWLWEKYGLRPDQWADYRALTGDESDNLPGVKGIGEKTARKLLEEWGSLEALLKNLDRLKPAIREKILAHMDDLKLSWDLAKVRTDLPLEVDFAKRREPDRERLRAFLERLEFGSLLHEFGLLESPNALEEAPWPPPEAAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEEIARLEAEVFRLAGHPFNLNSRDQLERVLFDELGLPAIGKTKKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDLIHPRTGRLHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQIELRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINFGVLYGMSAHRLSQELAIPYEEAQAFIERYFQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVRKAAERMAFNMPVQGTAADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWLSAKE。

Claims

1. A mutant Taq DNA polymerase, characterized in that, The amino acid sequence of the mutant Taq DNA polymerase corresponds to the sequence shown in SEQ ID NO: 4 and has the following multiple mutation sites: D58E, K292N, G304A, E507K, and E742K.

2. The mutant Taq DNA polymerase according to claim 1, wherein The amplification rate of the mutant Taq DNA polymerase is at least 1.5 times that of the wild-type Taq DNA polymerase; preferably at least 2 times; preferably at least 3 times; preferably at least 4 times.

3. A polynucleotide molecule, characterized in that, The sequence of the polynucleotide molecule corresponds to the nucleotide sequence shown in SEQ ID NO: 3, and the polynucleotide molecule encodes the mutant Taq DNA polymerase according to claim 1.

4. A recombinant vector, characterized in that, The vector contains the polynucleotide molecule according to claim 3.

5. A host cell, characterized in that, The host cell contains the vector according to claim 4 or the polynucleotide molecule according to claim 3 is chromosomally integrated.

6. A rapid PCR amplification kit, characterized in that, The kit contains the mutant Taq DNA polymerase according to claim 1.

7. A rapid PCR amplification kit according to claim 6, characterized in that, The kit further includes 5×Fast buffer, and the pH value of the 5×Fast buffer is 9.0 - 9.6; The 5×Fast buffer includes a buffer pair, a monovalent cation, an enhancer, and a surfactant.

8. A rapid PCR amplification kit according to claim 7, wherein The buffer pair is Tris-HCl; The monovalent cation includes NH4 + , K + One of them; The enhancer includes BSA and TMAC; The surfactant includes any one of Tween 20, CA-630, and Triton X-100.

9. A rapid PCR amplification kit according to claim 6, characterized in that, The kit further includes at least one of dNTP mixture, primer pair, Mg 2+ and water.

10. A method for rapid PCR amplification, characterized in that, The method includes performing PCR amplification using the 5×Fast buffer in the kit according to claim 7 and the mutant Taq DNA polymerase according to claim 1.

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