A DNA polymerase mutant, preparation method and application thereof

Abstract

This invention belongs to the field of biotechnology, specifically relating to a DNA polymerase mutant, its preparation method, and its application. The DNA polymerase mutant has the amino acid sequence shown in SEQ ID NO.1 and the nucleotide sequence shown in SEQ ID NO.2. This invention effectively solves the deficiency of existing technologies in the lack of highly resistant DNA polymerases tolerant to complex samples, enabling direct PCR amplification of whole blood, oral swabs, and bacterial culture samples without dilution or DNA extraction and purification.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a DNA polymerase mutant, its preparation method and application, and more specifically to an Escherichia coli recombinant expression DNA polymerase fusion, particularly to a high-fidelity, nucleic acid extraction-free (hereinafter referred to as PCR direct amplification) Pfu-Sso7d-6His DNA polymerase mutant, its preparation method and application. Background Technology

[0002] DNA polymerases are widely used enzymes in molecular biology, especially in polymerase chain reaction (PCR), where the quality and efficiency of the DNA polymerase determine the success of the experiment. Traditional Pfu DNA polymerase has high fidelity but relatively low processability, resulting in lower amplification efficiency compared to some other polymerases (such as Taq DNA polymerase). To address this issue, researchers created the Pfu-Sso7d fusion protein by fusing Sso7d (a DNA-binding protein from Sulfolobus solfataricus) with Pfu. Pfu, as a high-fidelity DNA polymerase, is heat-resistant; therefore, its fusion with Sso7d further enhances the stability and DNA-binding capacity of the entire enzyme complex in PCR reactions. Studies have shown that after 60 minutes of heat destruction at 98°C, Pfu-Sso7d retains 50%-60% activity, while Pfu completely loses its activity. Sso7d is a thermostable protein that maintains its DNA-binding capacity even at high temperatures. This characteristic is crucial for processes requiring high temperatures, such as PCR, as it helps the polymerase maintain effective binding to DNA at high temperatures.

[0003] The linker between Pfu-Sso7d is crucial because it relates to the steric hindrance effect between them. A linker that is too rigid provides stable and well-isolated fusion protein structures but limits function and reduces activity; a linker that is too flexible allows for dynamic freedom and folding-friendly fusion proteins, but results in a loose structure and low efficiency; positively charged linkers attract DNA and are friendly to low-concentration templates, thus increasing sensitivity, but leading to severe non-specific binding; negatively charged linkers repel DNA, preventing fusion protein structural collapse and aggregation that results in loss of activity; hydrophobic linkers stabilize the fusion protein structure and prevent aggregation / misfolding. These linker combinations affect the inhibitory resistance of Pfu-Sso7d; therefore, a suitable linker is essential for nucleic acid extraction-free, high-fidelity DNA polymerase mutants.

[0004] Table 1 Performance analysis of different types of linkers on DNA polymerase

[0005] Direct amplification (DPCR) technology for samples such as whole blood, oral swabs, and bacterial cultures is being widely applied in scenarios requiring rapid, efficient, and low-cost acquisition of genetic information. Highly resistant DNA polymerases capable of simultaneously tolerating inhibitors in these three types of complex samples have become key tools for advancing molecular detection and automating its implementation.

[0006] Compared to "direct amplification without diluent" and "direct amplification after dilution" and "traditional nucleic acid extraction followed by amplification", the core difference between whole blood, oral swabs and bacterial solutions lies in the trade-off between ease of operation, detection speed and amplification stability: direct sample addition is the fastest and simplest, especially suitable for grassroots screening and home self-testing, but it has extremely high requirements for the reagent's anti-inhibition ability, especially the enzyme's anti-interference performance.

[0007] Currently, there are very few DNA polymerases on the market that can simultaneously perform direct PCR amplification of whole blood, oral swabs, and bacterial samples without dilution or cell lysis buffer. This is primarily because these three types of samples have vastly different physical structures, chemical matrices, and inhibitor profiles, placing conflicting and extreme demands on the polymerase's tolerance, reaction system compatibility, and nucleic acid release mechanisms. To achieve true "instant detection," DNA polymerases and their accompanying buffer systems must simultaneously overcome the following technological hurdles: Whole blood: Ideally, cells should be gently lysed via PCR pre-denaturation (95°C) to avoid the excessive release of strong inhibitors such as hemoglobin and IgG. Oral swabs: Epithelial cell membranes are stable and highly keratinized; heating alone is insufficient to fully lyse the membrane, usually requiring the assistance of proteinase K or detergents. Bacterial culture: Cell walls are thick and dense, requiring strong lysis (e.g., lysozyme, mechanical grinding); otherwise, DNA cannot be exposed. If the polymerase system contains strong lysis components, it will exacerbate the release of inhibitors from whole blood; if not, insufficient DNA release from bacterial culture and oral cells will occur. Summary of the Invention

[0008] To address the shortcomings and deficiencies of the existing technologies, the present invention aims to provide a recombinant DNA polymerase mutant of Escherichia coli (abbreviated as PSUU-2G1K), which is a fusion protein of high-fidelity DNA polymerase (Pfu) and thermophilic bacteria Sso7d, in order to enable the DNA polymerase mutant to be directly amplified from whole blood (EDTA-K2) (without dilution buffer), directly amplified from oral swabs (without dilution buffer), and directly amplified from bacterial culture (without dilution buffer).

[0009] To achieve the objective of this invention, the inventors provide the following technical solution: The first objective of this invention is to provide a DNA polymerase mutant, the amino acid sequence of which is shown in SEQ ID NO.1.

[0010] The inventors have discovered that the present invention provides a recombinant expression of a DNA polymerase mutant in *E. coli* (abbreviated as PSUU-2G1K). This mutant is a fusion protein of high-fidelity DNA polymerase (Pfu) and thermophilic bacteria Sso7d. Verification has shown that the DNA polymerase mutant can achieve direct amplification of whole blood (EDTA-K2) (without dilution buffer), direct amplification of oral swabs (without dilution buffer), and direct amplification of bacterial cultures (without dilution buffer). The fusion protein of this invention innovatively uses a specially screened and optimized peptide linker (see SEQ ID NO.1 for details). Pfu and Sso7d linked by this linker exhibit excellent direct amplification performance. Verification has shown that the DNA polymerase mutant can increase the blood tolerance of EDTA-K2 whole blood samples to 44% (volume ratio, see [link to SEQ ID NO.1] for details). Figure 9 Oral swab tolerance increased to 44% (volume ratio, see details). Figure 7 The bacterial tolerance was increased to 44% (volume ratio, see details). Figure 10 All samples were direct amplifications without any dilution, and the human MTHFR gene polymorphism detection kit (fluorescent PCR-melting curve method) was used to test 12 whole blood samples and 12 oral swabs (see details). Figure 18 , 19 All of them could be amplified normally and the genotyping was completely correct, further demonstrating that the present invention has excellent direct amplification performance.

[0011] The second objective of this invention is to provide a method for preparing a DNA polymerase mutant, comprising the following steps: (1) Construction of DNA polymerase mutants: The Pfu DNA polymerase gene was fused with the Sso7d gene (Pfu-Sso7d), and the resulting recombinant plasmid (PSUU-2G1K) was constructed into the pET-26b(+) vector. A 6×His tag was added to the C-terminus of the fusion protein. The amino acid sequence is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2. (2) Gene cloning and expression: The DNA polymerase mutant recombinant plasmid constructed in step (1) was transformed into Escherichia coli BL21(DE3) cells to induce protein expression and obtain DNA polymerase mutant bacterial culture. (3) Protein purification: The DNA polymerase mutant bacterial culture obtained in step (2) was subjected to heat treatment, precipitation, resuspension and dissolution of the precipitate, affinity chromatography purification, and gradient elution to obtain the DNA polymerase mutant (abbreviated as PSUU-2G1K).

[0012] A third objective of this invention is to provide a nucleic acid molecule encoding a DNA polymerase mutant as described above, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0013] A fourth objective of this invention is to provide a recombinant vector containing the gene encoding the aforementioned DNA polymerase mutant. More preferably, the recombinant vector uses the pET-26b(+) vector as the expression vector.

[0014] A fifth objective of this invention is to provide the application of the aforementioned DNA polymerase mutant in the preparation of nucleic acid amplification products. More preferably, the nucleic acid amplification products include reagents and / or kits for PCR amplification. Even more preferably, they include reagents and / or kits for PCR amplification of whole blood samples or samples containing whole blood; reagents and / or kits for PCR amplification of oral swab samples or samples containing oral swab samples; and reagents and / or kits for PCR amplification of bacterial culture samples or samples containing bacterial culture.

[0015] The sixth objective of this invention is to provide the application of the aforementioned DNA polymerase mutant in PCR amplification of whole blood samples or samples containing whole blood.

[0016] The seventh objective of this invention is to provide the application of the aforementioned DNA polymerase mutant in PCR amplification of oral swab samples or samples containing oral swabs.

[0017] The eighth objective of this invention is to provide the application of the aforementioned DNA polymerase mutant in PCR amplification of bacterial culture samples or samples containing bacterial culture.

[0018] In a preferred embodiment of the present invention: The preparation method of the high-fidelity DNA polymerase mutant (PSUU-2G1K) in this invention mainly includes construction, expression, and purification, comprising the following steps: (1) Method for constructing high-fidelity DNA polymerase mutants: Pfu DNA polymerase was fused with Sso7d DNA-binding protein from *Sulfolobus solfataricus*, and the resulting gene was cloned into the pET-26b(+) vector. The recombinant plasmid PSUU-2G1K expressing the Pfu-Sso7d fusion protein was successfully constructed. A 6×His tag was introduced at the C-terminus of the fusion protein for subsequent affinity chromatography purification.

[0019] Nucleic acid and amino acid sequences: Sso7d protein was added to the C-terminus of Pfu and linked with a suitable linker (i.e., the part marked in green in SEQ ID NO.2) to maintain reasonable charge and flexibility of the fusion protein. The final fusion protein was designed as Pfu-linker-Sso7d-His-tag (nucleotide sequence see SEQ ID NO.2, amino acid sequence see SEQ ID NO.1).

[0020] (2) Gene cloning and expression: The constructed high-fidelity DNA polymerase mutant recombinant plasmid was transformed into Escherichia coli BL21(DE3) cells and cultured in LB medium. Isopropyl-β-D-thiogalactoside (IPTG) induced protein expression.

[0021] Optimize expression conditions (such as temperature, inducer concentration, induction time, etc.) to obtain high-fidelity DNA polymerase mutant bacterial culture.

[0022] (3) Protein purification: Cell lysis: E. coli was lysed using an ultrasonic disruption method to obtain a crude extract. The lysis buffer was Buffer A.

[0023] Heat treatment to remove heat-sensitive proteins: Heat treatment at 85℃ for 30 minutes, followed by centrifugation to remove proteins.

[0024] Ammonium sulfate precipitation of heat-resistant proteins (supernatant after heat treatment): The final concentration of ammonium sulfate in the supernatant is 300 g / L.

[0025] Resuspend and dissolve the precipitate: Use Buffer B.

[0026] Affinity chromatography purification: Affinity chromatography was performed using a nickel-NTA agarose resin. The His tag binds to nickel ions on the resin, and the target protein is specifically adsorbed. The loading solution was Buffer B containing the target protein (see Table 4 for details).

[0027] The fusion protein was eluted using a gradient elution buffer containing imidazole to determine the final washing buffer and elution buffer. The washing buffer was 1×PBS + 20mM imidazole (see Table 8 for details), and the elution buffer was 1×PBS + 500mM imidazole (see Table 9 for details).

[0028] Collect the eluent, verify the purity and concentration of the target elution peak using SDS-PAGE, and dialyze the high-concentration elution peak into high-fidelity DNA polymerase preservation solution using a dialysis bag.

[0029] Compared with the prior art, the advantages of the present invention are: (1) It can simultaneously perform direct amplification of whole blood, oral swabs, and bacterial culture without diluent. Clinically, the common practice is to use an automated nucleic acid extractor in conjunction with PCR amplification reagents. This approach is highly automated, convenient, and fast, but it also has obvious drawbacks. Automated nucleic acid extractors cost 50,000 to 100,000 yuan each, which is expensive. If a project is to be carried out, it is necessary to consider the automated nucleic acid extractor, the reagents and consumables that come with it. The cost of sample extraction is even higher than that of PCR amplification reagents. Direct amplification can solve this problem.

[0030] The DNA polymerase in this invention has strong anti-interference ability and can resist common PCR inhibitors such as heme in blood, polysaccharides and proteins in oral samples, and metabolites in bacterial cultures, ensuring stable amplification even in low-quality samples and improving the usability of clinical samples.

[0031] This invention eliminates the need for DNA extraction and purification steps, allowing direct amplification of samples such as whole blood and oral swab bacterial solutions. The entire process is reduced from several hours to tens of minutes, making it particularly suitable for scenarios requiring rapid results, such as newborn screening and rapid infectious disease testing.

[0032] Traditional extraction processes are prone to introducing exogenous DNA contamination or causing sample loss. Direct amplification technology reduces operational steps, lowers the probability of human error and cross-contamination, improves batch-to-batch repeatability, and is more suitable for automated and high-throughput detection.

[0033] Direct summarization results are more reliable and avoid false positives / false negatives.

[0034] (2) Fast and high fidelity This invention utilizes a specially designed linker to increase the Sso7d protein, thereby improving the process of Pfu DNA polymerase and thus enhancing PCR amplification efficiency, particularly in terms of extension speed and amplification sensitivity. The extension speed for short fragments is 0.71 S / kb (for short fragments <3000 base pairs, see details). Figure 11 ), long segments (13,000 base pairs, see details) Figure 1233S / kb. High-fidelity rapid direct-amplification PCR enzymes have not only improved the accuracy of detection in clinical applications, but also greatly shortened the detection time. They are an indispensable tool in clinical gene testing, pathogen detection, and genomics research.

[0035] Pfu DNA polymerase, widely recognized as a high-fidelity DNA polymerase, possesses 3'→5' exonuclease proofreading activity and exhibits a significantly lower amplification error rate than ordinary Taq DNA polymerase. It is widely used in applications requiring high amplification accuracy, such as gene cloning, sequencing, and mutation detection. This invention fuses a specific DNA-binding domain into Pfu DNA polymerase, preserving its inherent characteristics of high-fidelity amplification, low mismatch rate, and excellent amplification accuracy without altering the core catalytic structure and proofreading function of the Pfu enzyme. Simultaneously, the introduced DNA-binding domain enhances the enzyme's binding affinity to template DNA, improving amplification efficiency, reaction sensitivity, and tolerance to inhibitors in complex samples. Through this domain fusion strategy, this invention achieves synergistic optimization of amplification performance and high-fidelity characteristics, enabling the fusion enzyme to combine the high amplification accuracy of Pfu enzyme with enhanced template binding ability, meeting the needs of molecular diagnostics, gene detection, and scientific research applications for high-fidelity amplification enzymes.

[0036] (3) Can be used in fluorescence PCR-melting curve method The mechanism of Taq DNA polymerase-mediated quantitative PCR (such as the TaqMan method) is as follows: Taq polymerase has 5'→3' exonuclease activity, which hydrolyzes the probe bound to the template during DNA strand elongation, releasing a fluorescent signal. The DNA polymerase mutant in this invention does not have 5'→3' exonuclease activity, but it does have 3'→5' exonuclease activity, possessing the ability to correct mismatched bases. Fluorescent PCR-melting curve analysis (especially high-resolution melting curve analysis, HRM) often introduces mismatched bases to reduce and differentiate the Tm values ​​of different genotypes, thereby achieving highly sensitive detection of SNP sites, mutations, or methylation states. Therefore, the inventors used a human MTHFR gene polymorphism detection kit (fluorescent PCR-melting curve method, with mismatched bases) to test 12 whole blood samples and 12 oral swabs (see details). Figure 13-19 All samples were able to amplify normally and the genotyping was completely correct, further demonstrating that the present invention has excellent direct amplification performance and wide applicability.

[0037] (4) This invention has strong practical value. Potential applications include, but are not limited to: • PCR amplification: Suitable for high-fidelity PCR amplification, especially for amplification of long fragments with high GC content.

[0038] • Genetic engineering: The amplification step used in gene editing and DNA repair.

[0039] • Biopharmaceuticals: Can be used in the production of recombinant proteins, especially when used in combination with other enzymes.

[0040] • Fluorescent PCR-Melting Curve Method: High-fidelity rapid enzymes are mainly used in the fluorescent PCR-melting curve method to improve the accuracy and speed of amplification, and are especially suitable for melting curve analysis where high amplification specificity is required.

[0041] In melting curve analysis, especially when using the SYBR Green dye method, non-specific amplification (such as primer dimers or non-target products) can interfere with the interpretation of melting peaks, leading to misinterpretations. High-fidelity DNA polymerases possess 3'→5' exonuclease proofreading activity, which can significantly reduce amplification error rates and improve product accuracy, thereby ensuring clear melting curve peaks and stable Tm values, and enhancing the reliability of applications such as genotyping and mutation detection. Attached Figure Description

[0042] Figure 1 This is a portion of the nucleotide sequence encoding the DNA polymerase mutant of this invention, and... Figure 2 The nucleotide sequences form the complete sequence shown in SEQ ID NO.2.

[0043] Figure 2 This invention provides a portion of the nucleotide sequence encoding a DNA polymerase mutant, and... Figure 1 The nucleotide sequences form the complete sequence shown in SEQ ID NO.2. In the figure: the bold underline is PSUU-2G1K, the yellow and pink areas are restriction enzyme sites, the green area is the mutation site, and the rest is pET-26b(+).

[0044] Figure 3 This is an SDS-PAGE result of the DNA polymerase mutant of this invention. In the figure: the DNA polymerase mutant is the purified PSUU-2G1K mutant protein, and the control protein is bovine serum albumin (BSA).

[0045] Figure 4 This is a comparison of the direct amplification performance of the DNA polymerase mutant of this invention (PSUU-2G1K) with commercially available products. The results of the first round of screening show the direct amplification of oral swabs, whole blood samples, bacterial cultures, and plasmids near the detection limit of NEB Q5® Blood Direct 2×Master Mix.

[0046] Figure 5 This is a comparison of the direct amplification performance of the DNA polymerase mutant of this invention (PSUU-2G1K) with commercially available products. The results of the first round of screening show the direct amplification of PSUU-2G1K in oral swabs, whole blood samples, bacterial cultures, and plasmids near the detection limit.

[0047] Figure 6 This is a comparison of the direct amplification performance of the DNA polymerase mutant (PSUU-2G1K) of this invention with commercially available products. The results of the first round of screening show direct amplification of oral swabs, whole blood samples, bacterial cultures, and plasmids near the detection limit using Probe 2×qPCR Mix (Takara) and NEB Hemo KlenTaq® blood samples.

[0048] Figure 7 This is a comparison of the direct amplification performance of the DNA polymerase mutant (PSUU-2G1K) of this invention with commercially available products. The results of the second round of screening show the oral swab direct amplification tolerance of NEB Q5® Blood Direct 2×Master Mix and PSUU-2G1K.

[0049] Figure 8 This is a comparison of the direct amplification performance of the DNA polymerase mutant of this invention (PSUU-2G1K) with commercially available products. The whole blood direct amplification tolerance graph of NEB Q5® Blood Direct 2×Master Mix is ​​shown in the second round of screening results.

[0050] Figure 9 This is a comparison of the direct amplification performance of the DNA polymerase mutant of this invention (PSUU-2G1K) with commercially available products. The whole blood direct amplification tolerance graph of PSUU-2G1K is shown in the second round of screening results.

[0051] Figure 10 This is a comparison of the direct amplification performance of the DNA polymerase mutant of this invention (PSUU-2G1K) with commercially available products. The results of the second round of screening show the direct amplification tolerance of PSUU-2G1K and NEB Q5® Blood Direct 2×Master Mix.

[0052] Figure 11 This is an electrophoresis image (0.8% agarose gel electrophoresis image) of the DNA polymerase mutant (PSUU-2G1K) of this invention, with extension speeds set to 2s, 5s, 10s, 20s, 30s, 40s, 60s, 80s and 7min. The control is Pfu high-fidelity DNA polymerase.

[0053] Figure 12 This is an electrophoresis image (0.8% agarose gel electrophoresis image) of the amplified fragment length of the DNA polymerase mutant (PSUU-2G1K) of this invention. The long fragment amplification test showed amplified fragments of 7k, 9k, and 13k respectively.

[0054] Figure 13This is a fluorescence PCR-melting curve of the DNA polymerase mutant (PSUU-2G1K) of the present invention for testing the C677T of the MTHFR gene (typical CC-type melting peak).

[0055] Figure 14 This is a fluorescence PCR-melting curve (CT-type melting peak) of the DNA polymerase mutant (PSUU-2G1K) of the present invention for testing the C677T of the MTHFR gene.

[0056] Figure 15 This is a fluorescence PCR-melting curve diagram of the DNA polymerase mutant (PSUU-2G1K) of the present invention for testing the C677T of the MTHFR gene (melting peak of TT genotype).

[0057] Figure 16 This is a fluorescence PCR-melting curve diagram of the C677T of the MTHFR gene tested in this invention (melting peak of the negative control).

[0058] Figure 17 This is a fluorescence PCR-melting curve diagram of the C677T of the MTHFR gene tested in this invention (melting peak of the positive control).

[0059] Figure 18 This is a direct dilation curve diagram of 12 oral swabs from this invention.

[0060] Figure 19 This is a direct amplification curve of whole blood in 12 cases according to the present invention. Detailed Implementation

[0061] The present invention will be described in more detail below with reference to the embodiments. It should be understood that the implementation of the present invention is not limited to the embodiments below, and any modifications and / or alterations made to the present invention will fall within the protection scope of the present invention.

[0062] In this invention, unless otherwise specified, all parts and percentages are by weight, and all equipment and raw materials are commercially available or commonly used in the industry. Unless otherwise specified, the methods used in the embodiments are general techniques in the art.

[0063] Some key reagents and preparation methods described in the embodiments of this invention are as follows:

[0064]

[0065]

[0066]

[0067] Example 1: Acquisition and Detection Analysis of DNA Polymerase Mutant Protein 1. A method for obtaining a mutant DNA polymerase protein, mainly including gene construction, protein expression and purification, comprising the following steps: (1) Construction of recombinant expression vector pET-26b(+) was selected as the prokaryotic expression vector. Based on the designed fusion protein structure, the gene fragments encoding Pfu DNA polymerase and Sso7d DNA-binding protein were synthesized to construct a recombinant expression plasmid. To facilitate subsequent protein purification and detection, a 6×His tag sequence was introduced at the C-terminus of the fusion protein. After construction, a recombinant expression vector expressing Pfu-Sso7d fusion DNA polymerase was obtained.

[0068] Among them, Pfu DNA polymerase, as a high-fidelity DNA polymerase, provides polymerase activity and proofreading function, while Sso7d, as a small DNA-binding protein derived from thermophilic archaea, can enhance the binding ability between polymerase and template DNA, thereby improving amplification efficiency and amplification performance in complex samples.

[0069] (2) Expression of fusion protein The recombinant plasmid was transformed into the Escherichia coli BL21(DE3) expression host.

[0070] On the first day of cultivation, a single colony was picked and inoculated into LB medium (formula shown in Table 10) for small-scale overnight cultivation to obtain activated seed culture.

[0071] On the second day of cultivation, the seed culture was inoculated into the induction medium at an inoculation ratio of approximately 1% and cultured further. When the cells reached the logarithmic growth phase and the OD600 of the culture reached approximately 0.9, IPTG was added to induce expression, with a final IPTG concentration of 0.5 mM (formulation shown in Table 2). After adding the inducer, the culture temperature was lowered to 25°C and cultured overnight to allow the fusion protein to be fully expressed under lower temperature conditions, thereby improving the soluble expression level of the target protein.

[0072] (3) Collection of bacterial cells On the third day of culture, the induced bacterial cells were collected by centrifugation, the supernatant culture medium was discarded, and the recombinant bacterial cell precipitate containing the target fusion protein was obtained for subsequent protein extraction and purification.

[0073] (4) Cell lysis and preparation of crude extract The collected bacterial cells were resuspended in Buffer A lysis buffer (composition shown in Table 3), and the cells were lysed using sonication to release the intracellularly expressed fusion protein into the solution. After lysis, cell debris and unlysed cells were removed by centrifugation to obtain a crude extract containing the target protein.

[0074] (5) Purification of fusion protein To obtain high-purity fusion DNA polymerase, the crude extract was sequentially purified by heat treatment, ammonium sulfate precipitation, and nickel affinity chromatography. a) Heat treatment to remove heat-sensitive proteins Taking advantage of the excellent thermal stability of Pfu DNA polymerase, which is derived from thermophilic archaea, the crude extract was heat-treated. The crude extract was placed at 85℃ for 30 min, causing most of the E. coli proteins derived from the host bacteria to denature and precipitate, while the target fusion protein remained soluble.

[0075] After heat treatment, centrifuge at 12,000 rpm for 15 min, discard the precipitate, and collect the supernatant. This step effectively removes a large amount of heat-labile host proteins, improving subsequent purification efficiency.

[0076] b) Ammonium sulfate precipitates heat-resistant proteins Solid ammonium sulfate was slowly added to the supernatant after heat treatment for salting-out enrichment. Continuous stirring was maintained during the addition process to prevent localized protein denaturation caused by excessively high ammonium sulfate concentrations. The ammonium sulfate addition amount was 300 g / L of supernatant.

[0077] Salting out enriches the target protein in the precipitate, thereby achieving further concentration and preliminary purification.

[0078] c) Precipitation resuspension After collecting the ammonium sulfate precipitate, the protein precipitate was resuspended and fully dissolved in Buffer B (composition shown in Table 4) to obtain a protein sample suitable for subsequent chromatographic purification.

[0079] d) Ni-NTA affinity chromatography purification Ni-NTA agarose nickel affinity chromatography was used for purification. Because the fusion protein carries a 6×His tag at its C-terminus, it can specifically bind to nickel ions on the resin, while most other proteins cannot bind effectively, thus achieving the separation of the target protein from other proteins.

[0080] Protein samples dissolved in Buffer B were loaded into an equilibrated Ni-NTA affinity column. After loading, the column was first washed with a wash buffer containing a low concentration of imidazole to remove non-specifically adsorbed proteins. The wash buffer was 1×PBS + 20 mM imidazole (composition shown in Table 8).

[0081] Elution was then performed using an elution buffer containing a high concentration of imidazole. Imidazole competes with the His tag for binding to nickel ions, thereby causing the target fusion protein to dissociate from the resin and elute. The elution buffer was 1×PBS + 500 mM imidazole (composition shown in Table 9).

[0082] Each eluted fraction was collected and analyzed separately.

[0083] (6) Protein analysis and preservation SDS-PAGE was used to analyze each elution fraction, and the target elution peak was determined based on the position, purity, and concentration of the target protein band. Elution fractions with high target band concentration and good purity were selected for merging.

[0084] The combined target proteins were replaced with buffer (Buffer C, composition shown in Table 5) using a dialysis bag to gradually remove imidazole and other small molecule impurities, and finally dialyzed into high-fidelity DNA polymerase preservation solution (composition shown in Table 12).

[0085] After dialysis, a purified Pfu-Sso7d fusion DNA polymerase preparation is obtained, which can be used for subsequent enzymatic property evaluation, PCR amplification performance testing, and direct amplification performance verification of complex samples.

[0086] The amino acid sequence of the DNA polymerase mutant is shown in SEQ ID NO.1.

[0087] SEQ ID NO.1: Amino acid sequence: pET-26b(+)-PSUU-2G1K, 849aa.

[0088] MILDVDYITEEGKPVIRLFKKENGKFKIEHDRTFRPYIYALLRDDSKIEEVKKITGERHGKIVRIVDVEKVEKKFLGKPITVWKLYLEHPQDVPTIREKVREHPAVVDIFEYDIPFAKRYLIDKGLIPMEGEEELKILAFDIETLYHEGEEFGKGPIIMISYADENEAKVITWKNIDLPYVEVVSSEREMIKRFLRIIREKDPDIIVTYNGDPFDFPYLAKRAEKLGIKLTIGRDGSEPKMQRIGDMTAVEVKGRIHFDLYHVITRTINLPTYTLEAVYEAIFGKPKEKVYADEIAKAWESGENLERVAKYSMEDAKATYELGKEFLPMEIQLSRLVGQPLWDVSRSSTGNLVEWFLLRKAYERNEVAPNKPSEEEYQRRLRESYTGGFVKEPEKGLWENIVYLDFRALYPSIIITHNVSPDTLNLEGCKNYDIAPQVGHKFCKDIPGFIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYAKARWYCKECAESVTAWGRKYIELVWKELEEKFGFKVLYIDTDGLYATIPGGESEEIKKKALEFVKYINSKLPGLLELEYEGFYKRGFFVTKKRYAVIDEEGRVITRGLEIVRRDWSEIAKETQARVLETILKHGDVEEAVRIVKEVIQKLANYEIPPEKLAIYEQITRPLHEYKAIGPHVAVAKKLAAKGVKIKPGMVIGYIVLRGDGPISNRAILAEEYDPKKHKYDAEYYIENQVLPAVLRILEGFGYRKEDLRYQKTRQVGLTSWLNIKKSGTHKHKHGTHMATVKFKYKGEEKEVDISKIKKVWRVGKMISFTYDEGGGKTGRGAVSEKDAPKELLQMLEKQKK The nucleotide sequence encoding the DNA polymerase mutant is shown in SEQ ID NO. 2.

[0089] SEQ ID NO.2: DNA sequence: pET-26b(+)-PSUU-2G1K (the bold underline is PSUU-2G1K, the yellow and pink areas are restriction enzyme sites, the green area is the mutation site, and the rest is pET-26b(+)) (see...) Figure 1 , Figure 2 ).

[0090] 2. Performance Analysis and Activity Testing The predicted information for the fusion protein-DNA polymerase mutant in this invention is as follows: isoelectric point pH 8.48, relative molecular weight 99363.83 Da.

[0091] (1) To evaluate the expression and purification effect of the DNA polymerase mutant PSUU-2G1K of the present invention, the purified protein was analyzed by SDS-PAGE.

[0092] The purified DNA polymerase mutant protein sample was mixed with protein loading buffer and denatured, then loaded onto a polyacrylamide gel for electrophoresis. After electrophoresis, the gel was stained and destained, and the distribution of protein bands was observed.

[0093] The electrophoresis results were analyzed based on the theoretical molecular weight of the target protein. The results showed that a clear and specific protein band was observed at approximately 99.4 kDa, which is consistent with the theoretical molecular weight of the target DNA polymerase mutant, indicating that the target protein was successfully obtained.

[0094] The electrophoresis results were further analyzed using a gel imaging system, and protein purity was calculated based on the gray-scale integral value of the target band and the total protein band in the lane. The results showed that the purified DNA polymerase mutant PSUU-2G1K achieved a purity of over 95%, meeting the requirements for subsequent enzymatic performance evaluation and application research. Detailed results can be found in [link to detailed results]. Figure 3 .

[0095] (2) Performance Analysis A very high extension rate was detected when the DNA polymerase mutant purified according to this invention was used in a standard PCR reaction.

[0096] (2.1) Amplification rate To evaluate the amplification rate of the DNA polymerase mutant PSUU-2G1K of this invention, DNA fragments of different lengths were selected as amplification targets, corresponding amplification primers were designed, and purified PSUU-2G1K was used for amplification in a standard PCR reaction system. Different extension time conditions were set for PCR amplification of the target fragments, and nucleic acid electrophoresis was used to analyze the specificity and integrity of the amplification products. The results showed that under the reaction conditions of this embodiment, PSUU-2G1K could amplify approximately 2800 bp (2.8 kb) of the target fragment within a 2-second extension time, corresponding to an amplification rate of approximately 0.71 s / kb; simultaneously, it could amplify approximately 13000 bp (13 kb) of the target fragment within a 7-minute extension time, corresponding to an amplification rate of approximately 33 s / kb. Based on the test results of amplified fragments of different lengths, PSUU-2G1K exhibits high amplification efficiency in conventional PCR amplification applications, with an amplification rate reaching 5–15 s / kb. Related results are shown in [link to relevant results]. Figure 11 .

[0097] (2.2) Amplified fragment length To evaluate the long-fragment amplification capability of the DNA polymerase mutant PSUU-2G1K of this invention, DNA template regions of different lengths were selected as amplification targets, and corresponding amplification primers were designed. Purified PSUU-2G1K was added to a standard PCR reaction system for amplification, and the PCR products were detected by nucleic acid electrophoresis after amplification.

[0098] The long fragment amplification capability of PSUU-2G1K was evaluated by comparing the consistency between the actual amplified product and the theoretical target fragment size, as well as the integrity and specificity of the amplified band.

[0099] The long-fragment amplification capability of PSUU-2G1K was evaluated using self-designed amplification primers (see Table 19). Under the template, primers, and reaction conditions used in this example, PSUU-2G1K successfully amplified a target fragment up to 13000 bp (13 kb), indicating that the enzyme has good long-fragment amplification capability. Related results are shown in [Table 19]. Figure 12 It should be understood that the maximum fragment length that PCR can amplify is affected by various factors, including template quality, template complexity, primer design, reaction system composition, and amplification conditions. After optimizing these conditions, those skilled in the art can further evaluate the enzyme's ability to amplify longer DNA fragments.

[0100] Example 2: Determination of the anti-interference ability of the DNA polymerase mutant in this invention: direct amplification of whole blood, direct amplification of oral swabs, and direct amplification of bacterial solutions. Whole blood samples: To evaluate the tolerance of the DNA polymerase mutant PSUU-2G1K of this invention to PCR inhibitors in whole blood samples, direct amplification of whole blood was used for verification.

[0101] Human peripheral blood was collected intravenously into EDTA-K2 anticoagulant tubes. Immediately after collection, the tubes were gently inverted to mix, yielding EDTA-K2 anticoagulant whole blood samples. These whole blood samples were used directly as templates for PCR reactions without DNA extraction, lysis, or dilution.

[0102] The PCR reaction system is shown in Table 14, with a total reaction volume of 25 μL. Different volumes of whole blood sample were added according to preset proportions, and the remaining volume was made up with nuclease-free water. The final volume percentages of whole blood sample were set as follows: 2% (0.5 μL), 4% (1 μL), 8% (2 μL), 16% (4 μL), 24% (6 μL), 32% (8 μL), 40% (10 μL), and 44% (11 μL).

[0103] The PCR amplification program was performed according to Table 15: pre-denaturation at 98℃ for 2 min; followed by 40 cycles, including denaturation at 95℃ for 30 s, annealing at 55℃ for 30 s and extension at 72℃ for 30 s; and a final extension at 72℃ for 3 min after the cycle.

[0104] After amplification, the PCR products were detected by agarose gel electrophoresis. Obtaining amplified products of the expected size with clear bands was used as the criterion for successful amplification, to evaluate the tolerance of the DNA polymerase mutant to hemoglobin, immunoglobulins, lactoferrin, and other PCR-inhibiting components in whole blood samples.

[0105] The experimental results show that the DNA polymerase mutant PSUU-2G1K of this invention has excellent resistance to whole blood interference. Even with whole blood samples accounting for 44% of the total PCR reaction volume, the target amplification product can still be obtained, indicating that it can tolerate PCR inhibitors in high proportions of whole blood samples. Detailed results can be found in [link to detailed results]. Figure 9 .

[0106] Oral swabs (exfoliated cells from the oral mucosa): To evaluate the tolerance of the DNA polymerase mutant PSUU-2G1K of this invention to PCR inhibitors in oral swab samples, direct amplification of oral swabs was used for verification.

[0107] A sterile swab is inserted into the oral cavity and repeatedly scrapes the buccal mucosa (inner cheek) 15–20 times to collect exfoliated cells. After sampling, the swab head is placed in a sample tube containing sample preservation solution, and the tube is broken near the break point. The swab head is retained in the sample tube and sealed for preservation. In this embodiment, ddH2O is used as the sample preservation solution to increase the difficulty of sample tolerance evaluation.

[0108] The PCR reaction system is shown in Table 14, with a total reaction volume of 25 μL. Oral swab samples were added directly to the PCR reaction system as templates without nucleic acid extraction, lysis, or dilution. Final sample concentrations were set at 2% (0.5 μL), 4% (1 μL), 8% (2 μL), 16% (4 μL), 24% (6 μL), 32% (8 μL), 40% (10 μL), and 44% (11 μL), with the remaining volume brought to 25 μL using ddH2O.

[0109] PCR amplification conditions were performed according to Table 15: pre-denaturation at 98℃ for 2 min; followed by 40 cycles, including denaturation at 95℃ for 30 s, annealing at 55℃ for 30 s and extension at 72℃ for 30 s; and a final extension at 72℃ for 3 min after the cycle.

[0110] After amplification, the PCR products were detected by agarose gel electrophoresis. Obtaining amplification products of the expected size with clear bands was used as the criterion for successful amplification, and the tolerance of the DNA polymerase mutant to mucin, food residues, cell debris, and other PCR-inhibiting components in oral samples was evaluated accordingly.

[0111] The results show that the DNA polymerase mutant PSUU-2G1K of this invention exhibits excellent direct amplification capability from oral swabs. Under conditions requiring no nucleic acid extraction, no lysis treatment, and no dilution buffer, the target amplification product can still be obtained when the oral swab sample accounts for 44% of the total PCR reaction volume, indicating that the enzyme can tolerate high proportions of PCR inhibitors in oral samples. Detailed results can be found in [link to detailed results]. Figure 7 .

[0112] Determination of interference resistance by direct amplification of bacterial culture: To evaluate the tolerance of the DNA polymerase mutant PSUU-2G1K of the present invention to PCR inhibitors in bacterial culture samples, direct amplification of bacterial culture was used for verification.

[0113] The bacterial culture sample used in the experiment was an overnight culture of BL21(DE3) Escherichia coli (OD600≈3.0). Sterile glycerol was added to the culture to make the final concentration of glycerol 25%, resulting in a bacterial culture sample with an OD600 of approximately 1.5.

[0114] The PCR reaction system is shown in Table 14, with a total reaction volume of 25 μL. Bacterial culture samples were added directly to the PCR reaction system as templates without nucleic acid extraction, cell lysis, or dilution. Final bacterial culture concentrations were set at 2% (0.5 μL), 4% (1 μL), 8% (2 μL), 16% (4 μL), 24% (6 μL), 32% (8 μL), 40% (10 μL), and 44% (11 μL), with the remaining volume brought to 25 μL using ddH2O.

[0115] PCR amplification conditions were performed according to Table 15: pre-denaturation at 98℃ for 2 min; followed by 40 cycles, including denaturation at 95℃ for 30 s, annealing at 55℃ for 30 s and extension at 72℃ for 30 s; and a final extension at 72℃ for 3 min after the cycle.

[0116] After amplification, the PCR amplification products were detected by agarose gel electrophoresis. Obtaining amplification products of the expected size with clear bands was used as the criterion for successful amplification, and this was used to evaluate the tolerance of the DNA polymerase mutant to bacterial components, culture medium residues, glycerol, and other PCR inhibitors.

[0117] The results show that the DNA polymerase mutant PSUU-2G1K of this invention exhibits excellent direct amplification capability from bacterial cultures. Under conditions requiring no nucleic acid extraction, no lysis treatment, and no dilution buffer, the target amplification product can still be obtained when the bacterial culture sample accounts for 44% of the total PCR reaction volume, indicating that the enzyme can tolerate PCR inhibitors in a high proportion of bacterial culture samples. Detailed results can be found in [link to detailed results]. Figure 10 .

[0118]

[0119] Example 3: Fluorescent PCR-Melting Curve Method The PCR reaction system was prepared using the 2×PSUU reaction solution shown in Table 13, along with primers and probes provided by a commercially available human MTHFR gene polymorphism detection kit, and the sample to be tested. The sample to be tested included whole blood samples and oral swab samples; neither type of sample required nucleic acid extraction and could be used directly as amplification templates.

[0120] The negative control (NC) uses nuclease-free water instead of template in the reaction system to monitor for contamination in the amplification system (see negative control). Figure 16 ).

[0121] Positive controls are provided with the kit's positive control material or standard sample DNA with a genotype confirmed by a reference method (the reference method is the MTHFR C677T genotype confirmed by Sanger sequencing) to verify whether the amplification system and melting curve analysis process are normal (see positive control). Figure 17).

[0122] After the reaction system is prepared, it is placed in a real-time fluorescence PCR instrument for amplification and detection. The amplification program is set according to Table 16: (1) React at 25℃ for 5 min; (2) Pre-denaturation at 95℃ for 5 min; (3) Enter the amplification cycle, a total of 50 cycles, including: denaturation at 95℃ for 10 s; annealing at 54℃ for 15 s; extension at 68℃ for 25 s.

[0123] The fluorescence detection channel during the amplification stage was set to the Cy5 channel.

[0124] After amplification, melting curve analysis was performed. The melting curve program was set up as shown in Table 16: The temperature was maintained at 95℃ for 40 s; then at 40℃ for 3 min; subsequently, the temperature was increased from 40℃ to 70℃ at a rate of 0.06℃ / s. Fluorescence signals were continuously collected during the heating process, and melting curves and melting peak spectra were generated.

[0125] (3) Result interpretation Genotyping analysis was performed based on the amplification and melting curve results.

[0126] The negative control should have no obvious amplification curve and no specific melting peak; if the negative control shows an amplification signal or a specific melting peak, it indicates that there may be contamination in the experimental process, and the results of this experiment are invalid.

[0127] The positive control should obtain a normal amplification curve and show a characteristic melting peak consistent with its corresponding genotype, indicating that the detection system is operating normally.

[0128] Genotyping of the test samples is performed based on the position of the melting peak: CC-type samples show characteristic melting peaks of the wild type; TT-type samples show characteristic melting peaks of the mutant type; CT-type samples show melting peaks corresponding to both the wild type and the mutant type.

[0129] (4) Result verification The reaction system was prepared using the DNA polymerase mutant PSUU-2G1K of this invention, and 12 whole blood samples and 12 oral swab samples were tested. All samples yielded normal amplification curves and clearly identifiable melting peaks. The test results were compared with the genotyping results confirmed by a reference method. The results showed that the genotyping results of 24 samples were completely consistent (the reference method was the MTHFR C677T genotype confirmed by Sanger sequencing), accurately identifying the CC, CT, and TT genotypes at the MTHFR gene C677T locus.

[0130] The results showed that the DNA polymerase mutant of this invention is suitable for detecting the C677T site of the human MTHFR gene using fluorescent PCR-melting curve method, exhibiting good amplification performance and genotyping accuracy under both whole blood direct amplification and oral swab direct amplification conditions. Detection results are shown below. Figures 13-19 .

[0131]

[0132] Comparative Example 1: Direct expansion performance of DNA polymerase mutant PSUU-2G1K compared with commercially available products. In the field of molecular biology detection, the amplification efficiency and detection accuracy of PCR technology largely depend on the tolerance of DNA polymerase to PCR inhibitors in complex samples. Hemoglobin, immunoglobulins, and lactoferrin in whole blood samples, mucin and cell debris in oral swab samples, and culture medium components, cell debris, and metabolites in bacterial culture samples can all inhibit PCR amplification. Therefore, this embodiment uses direct amplification of whole blood, direct amplification of oral swabs, and direct amplification of bacterial culture as evaluation models to compare the anti-interference ability of the DNA polymerase mutant PSUU-2G1K of this invention with commercially available products NEB Q5® BloodDirect 2× Master Mix, NEB Hemo KlenTaq®, and TakaraProbe 2× qPCR Mix.

[0133] The same amplification system and amplification procedure were used in both the first and second rounds of screening.

[0134] (1) PCR reaction system The PCR reaction system is shown in Table 17, with a total reaction volume of 25 μL. The reaction system included 12.5 μL of 2×PSUU reaction solution, 0.5 μL of 10 μM upstream primer, 0.5 μL of 10 μM downstream primer, 0.5 μL of DNA polymerase mutant, 1 μL of the sample to be tested, and ddH2O to a final volume of 25 μL. The primer sequences used are shown in Tables 19-A, 19-B, and 19-C.

[0135] For the control group of commercially available products, the configuration should be carried out according to the recommended system in the instructions for each product, and the primers, templates and total volume should be kept consistent with those of the experimental group.

[0136] (2) PCR amplification program The PCR amplification conditions are shown in Table 18.

[0137] First, pre-denaturation was performed at 98℃ for 2 min; then 40 cycles were performed, each cycle consisting of: denaturation at 95℃ for 30 s; annealing at 55℃ for 30 s; extension at 72℃ for 30 s; after the cycle, final extension at 72℃ for 3 min was performed.

[0138] After the amplification reaction was completed, the PCR products were analyzed by agarose gel electrophoresis.

[0139] (3) Result determination Obtaining amplified products of the expected size with clear bands is used as the criterion for successful amplification.

[0140] As the proportion of samples added increases, the concentration of PCR inhibitors also increases. When a clear and specific amplification band can still be obtained at a certain concentration, the DNA polymerase is considered to be tolerant to the PCR inhibitor in the corresponding proportion of samples; when the amplification band is significantly weakened, non-specific amplification occurs, or there is no amplification product at all, the enzyme is considered to have reached its tolerance limit.

[0141] The anti-interference ability of different DNA polymerases was evaluated by comparing the amplification results under the same sample type and sample addition ratio.

[0142] (4) Sample type Whole blood samples were EDTA-K2 anticoagulated whole blood and added directly to the PCR reaction system without DNA extraction, lysis or dilution. Oral swab samples were collected from exfoliated cells of the buccal mucosa using sterile swabs and stored in ddH2O. They were then added directly to the PCR reaction system without nucleic acid extraction or dilution. The bacterial culture samples were prepared using an overnight culture of BL21(DE3) Escherichia coli and were directly added to the PCR reaction system without nucleic acid extraction or dilution.

[0143] By gradually increasing the volume ratio of samples in the PCR system, the anti-inhibition ability of each DNA polymerase under complex sample direct amplification conditions was evaluated, and the highest tolerable sample ratio was used as the evaluation index.

[0144] In the field of molecular biology detection, the efficiency and accuracy of PCR technology largely depend on the performance of DNA polymerase, especially its resistance to interference when faced with complex samples. Whole blood, oral swabs, and bacterial cultures are common sample types in clinical diagnosis, forensic identification, and microbial testing. Their complex composition, rich in heme, mucin, polysaccharides, and other PCR inhibitors, poses a severe challenge to the polymerase's tolerance. This comparative experiment will focus on the DNA polymerase mutant of this invention and products from internationally renowned manufacturers (NEB Q5® Blood Direct 2×Master Mix, NEB Hemo KlenTaq). ® A deep comparison of the anti-interference capabilities of blood-derived amplification and Probe 2×qPCR Mix (Takara) in three scenarios—direct amplification of whole blood, direct amplification of oral swabs, and direct amplification of bacterial culture—was conducted.

[0145] The NEB Q5® Blood Direct 2× Master Mix uses Q5® hot-start ultra-fidelity DNA polymerase, which has 3′→5′ exonuclease activity but lacks the 5′→3′ exonuclease activity to cleave TaqMan probes. Therefore, it cannot be used for probe-based qPCR (such as TaqMan assays). The Takara Probe 2× qPCR Mix uses antibody-modified hot-start Taq DNA polymerase, which, upon activation, possesses 5′→3′ exonuclease activity. This enzyme can recognize and cleave TaqMan probes bound to the target sequence, releasing a fluorescent signal. Therefore, it is specifically designed for probe-based qPCR and is suitable for highly sensitive and specific quantitative detection. NEB Q5 blood-derived direct amplification 2× premixed solution has significant advantages in whole blood direct amplification scenarios. Its high fidelity and long-fragment amplification capability make it suitable for fields with extremely high accuracy requirements, such as clinical gene diagnosis and forensic evidence identification. Probe2×qPCR Mix (Takara) performs well in scenarios such as oral swab testing and low-concentration bacterial culture quantitative analysis due to its good compatibility and sensitivity, making it more suitable for large-scale sample testing such as clinical screening and environmental monitoring. Hemo KlenTaq is a truncated version of... Taq DNA polymerase, lacking the first 280 amino acids. Hemo KlenTaq contains some mutations that allow it to tolerate inhibitors present in whole blood. This enzyme can directly amplify DNA from human and mouse whole blood samples. Hemo KlenTaq is tolerant to up to 20% whole blood in a 25µl reaction system (up to 30% whole blood in a 50µl reaction system). The PCR reaction systems (Table 17 PCR Reaction Systems) and PCR reaction conditions (Table 18 PCR Reaction Conditions) used in the first and second rounds of screening are as follows:

[0146] Round 1: The DNA polymerase mutant of this invention is compared with direct amplification products from international manufacturers, and the superior direct amplification products are selected for further comparison.

[0147] Three pairs of primers were designed for human-specific sequences, amplifying fragment sizes of 181bp, 458bp, and 206bp (see A, B, and C in Table 19). Human oral swabs and human EDTA-K2 whole blood were used as templates (the template was uniformly 4%, i.e., 1μL).

[0148] Since high-fidelity DNA polymerase has strong 3′→5′ exonuclease activity, in order to verify whether degenerate primers have a significant effect on high-fidelity DNA polymerase, a pair of degenerate primers (see D in Table 19) were designed for the Escherichia coli genome. The amplified fragment size was 469 bp, and bacterial culture was used as a template (the template content was uniformly 4%).

[0149] To validate samples near the detection limit (0.05 ng / reaction well), a pair of primers were designed to amplify fragments of 197 bp (see E in Table 19), and 0.05 ng / μL of pUC18 plasmid was used as a template (0.05 ng / reaction well).

[0150] The second round: Further evaluation of the direct amplification tolerance of the DNA polymerase mutant (PSUU-2G1K) from this invention with the excellent direct amplification products obtained in the first round of screening was conducted. Considering that the NEB Q5® Blood Direct 2× Master Mix is ​​2×, and primers and probes need to be added, and that plasma accounts for approximately 55% of the whole blood volume and blood cells approximately 45%, the actual whole blood template volume was increased to 44%. After the PCR reaction, the liquid volume in the reaction system was already very small, making it difficult to obtain a sufficient amount of liquid for identifying amplified bands. Therefore, the maximum template volume for whole blood samples, oral swabs, and bacterial culture samples was set to 44% (V / V), and the template volume gradients were set to 2% (0.5μL), 4% (1μL), 8% (2μL), 16% (4μL), 24% (6μL), 32% (8μL), 40% (10μL), and 44% (11μL) (V / V).

[0151] Results of the first round of testing (see details) Figure 4-6 ): Direct oral swab analysis: PSUU-2G1K, NEB Q5® Blood Direct 2×Master Mix, NEB HemoKlenTaq ® Both blood-derived amplification and Probe 2×qPCR Mix (Takara) were tolerant to 8% oral swabs and could correctly amplify the target bands of 181bp, 458bp, and 206bp. Whole blood direct amplification: Probe 2×qPCR Mix (Takara) could not tolerate 8% whole blood and failed to correctly amplify the target bands of 181bp, 458bp, and 206bp; NEB Hemo KlenTaq ® Blood-derived amplification can only partially amplify the target band (correctly amplifying 181bp and 206bp, but failing to amplify 458bp).

[0152] Direct amplification of bacterial culture: except for NEB Hemo KlenTaq ®While direct amplification of bacterial culture samples from the blood-derived amplification kit was unsuccessful (target fragment was 469bp), direct amplification of bacterial culture samples from PSUU-2G1K, NEB Q5® Blood Direct 2×Master Mix, and Probe2×qPCR Mix (Takara) was successful (target fragment was 469bp). Plasmid amplification near the detection limit: Except for PSUU-2G1K, which could normally amplify the pUC18 plasmid, NEB Q5® Blood Direct 2×Master Mix and NEBHemo KlenTaq... ® Both blood-derived amplification and Probe 2×qPCR Mix (Takara) failed to amplify normally.

[0153] Therefore, PSUU-2G1K and NEB Q5® Blood Direct 2×Master Mix were preferred for the second round of tolerance testing, while Probe2×qPCR Mix (Takara) was not used for the second round of tolerance testing. Whole blood direct amplification, oral swab direct amplification, and bacterial culture direct amplification were tested, with EDTA-K2 templates used for whole blood samples, oral swabs, and bacterial cultures. The amplified fragment sizes were 181bp (EDTA-K2 whole blood sample direct amplification, oral swab direct amplification) and 469bp (bacterial culture direct amplification), respectively.

[0154] The results of the second round of testing (see details) Figure 7-10 ): Oral swab sample volumes (by volume) were 2% (0.5 μL), 4% (1 μL), 8% (2 μL), 16% (4 μL), 24% (6 μL), 32% (8 μL), 40% (10 μL), and 44% (11 μL). The experimental results showed that the DNA polymerase mutant PSUU-2G1K and NEB Q5® Blood Direct 2×Master Mix of this invention were both tolerated in 44% oral swab samples (see detailed results). Figure 7 ).

[0155] Whole blood sample volumes (volume ratios) were 2% (0.5 μL), 4% (1 μL), 8% (2 μL), 16% (4 μL), 24% (6 μL), 32% (8 μL), 40% (10 μL), and 44% (11 μL). The experimental results show that the DNA polymerase mutant PSUU-2G1K in this invention can tolerate 44% whole blood sample volume (see detailed results). Figure 8 , 9 NEB Q5® Blood Direct 2×MasterMix is ​​tolerable with 32% whole blood.

[0156] The bacterial suspension volumes (volume ratios) were 2% (0.5 μL), 4% (1 μL), 8% (2 μL), 16% (4 μL), 24% (6 μL), 32% (8 μL), 40% (10 μL), and 44% (11 μL). The experimental results showed that the DNA polymerase mutant of this invention could tolerate 44% of the bacterial suspension sample (see detailed results). Figure 10 (Non-BQ5® Blood Direct 2×Master Mix can only tolerate 32% bacterial concentration), while NEB Q5® Blood Direct 2×Master Mix can only tolerate 32% bacterial concentration.

[0157] Evaluation of the extension rate and amplified fragment length of DNA polymerase mutant PSUU-2G1K in Comparative Example 2 (see details) Figure 11 , 12 ) To evaluate the amplification rate and long fragment amplification capability of the DNA polymerase mutant PSUU-2G1K of this invention, Pfu high-fidelity DNA polymerase was used as a control, and PCR amplification tests were performed using templates from different sources and amplification fragments of different lengths.

[0158] Amplification primers were designed based on publicly available sequences from the NCBI database. The primer sequences are shown in Table 19. (The table includes the primer sequences for reference only.) Primers A (181 bp), B (458 bp), and C (206 bp) were derived from human genome sequences, with templates being whole blood and oral swab samples, respectively. Primers D (469 bp) and I (T7) were derived from the Escherichia coli genome sequence, and the template was BL21(DE3) glycerol culture. Primer E (197 bp) was derived from the pUC18 plasmid sequence, and the template was pUC18 plasmid DNA; Primers F (approximately 7 kb), G (approximately 9 kb), and H (approximately 13 kb) are derived from λDNA sequences, with λDNA as the template.

[0159] (1) PCR reaction system The PCR reaction system is shown in Table 20. The total reaction volume is 25 μL, including: 12.5 μL of 2×PSUU reaction solution; 0.5 μL of 10 μM upstream primer; 0.5 μL of 10 μM downstream primer; 0.5 μL of DNA polymerase mutant; X μL of template; and ddH2O to bring the total volume to 25 μL.

[0160] The control group used an equal amount of Pfu high-fidelity DNA polymerase instead of PSUU-2G1K, while keeping the other components and reaction conditions the same.

[0161] (2) PCR amplification conditions The PCR amplification procedure is shown in Table 21.

[0162] First, pre-denaturation was performed at 98℃ for 2 min; then 40 cycles were performed, each cycle consisting of: denaturation at 95℃ for 30 s; annealing at 55℃ for 30 s; extension at 72℃ for 2–420 s; and final extension at 72℃ for 3 min after the cycle.

[0163] The extension time at 72℃ was adjusted according to the length of the target fragment to be amplified and the experimental purpose.

[0164] (3) Evaluation of amplification rate To evaluate the DNA polymerase extension rate, λDNA was used as a template, and target fragments of approximately 7 kb, 9 kb, and 13 kb were amplified using primers F, G, and H, respectively.

[0165] While keeping the other PCR conditions consistent, the extension time at 72℃ was set to 2 s, 5 s, 10 s, 20 s, 30 s, 40 s, 60 s, 80 s and 7 min, respectively, and the amplification of the target fragment under different extension times was compared.

[0166] After the PCR reaction, the amplification products were analyzed by agarose gel electrophoresis. Obtaining amplification products of the same size as the theoretical value and with clear bands was used as the criterion for successful amplification.

[0167] The extension rate of DNA polymerase was calculated based on the minimum extension time required for successful amplification and the corresponding amplified fragment length, and compared with that of Pfu high-fidelity DNA polymerase.

[0168] The results showed that the DNA polymerase mutant PSUU-2G1K of this invention exhibited high DNA strand elongation efficiency. Approximately 2.8 kb of the target fragment could be effectively amplified under 2 s elongation conditions; approximately 13 kb of the target fragment could be effectively amplified under 7 min elongation conditions, demonstrating excellent rapid amplification capability. Detailed results can be found in [link to detailed results]. Figure 11 .

[0169] (4) Evaluation of amplified fragment length To evaluate the long-fragment amplification capability of DNA polymerase, λDNA was used as a template, and DNA fragments of different lengths were amplified using primers F, G, and H, respectively.

[0170] After PCR amplification, the amplification results were analyzed by agarose gel electrophoresis, and the amplification ability of long fragments was evaluated based on the consistency between the amplified band size and the theoretical length, as well as the band integrity.

[0171] The results showed that the DNA polymerase mutant PSUU-2G1K of this invention could successfully amplify DNA fragments up to 13 kb in length, and the amplified products exhibited good specificity and integrity, indicating its excellent long-fragment amplification capability. Detailed results can be found in [link to detailed results]. Figure 12 .

[0172] It should be understood that the maximum fragment length that can be amplified by PCR is affected by a variety of factors such as template integrity, primer design, GC content, reaction system composition and amplification conditions. Therefore, the 13 kb amplification result obtained in the embodiments of the present invention does not constitute a limitation on the maximum amplification capacity of the enzyme.

[0173]

[0174] This invention utilizes a specially designed linker (the green portion in SEQ ID NO.2) to increase the Sso7d protein, thereby enhancing the process of PfuDNA polymerase and improving PCR amplification efficiency. The short fragment (2800bp, detailed results can be found in...) Figure 11-2 The extension rate of the 800bp amplified fragment was 0.71 s / kb (2800bp amplification in 2 seconds, calculated as 0.71 s / kb); while the extension rate of Pfu high-fidelity DNA polymerase was 14.29 s / kb (2800bp amplification in 40 seconds, calculated as 14.29 s / kb), 20 times faster than Pfu high-fidelity DNA polymerase. For the long fragment (12567bp, see detailed results...), see... Figure 12-13 (K amplified fragment) 33s / kb (12.567kb amplified in 7 minutes, calculated as 33s / kb).

[0175] Compared to the industry benchmark NEB Q5® Blood Direct 2×Master Mix, the DNA polymerase mutant PSUU-2G1K in this invention is comparable to NEB's Q5 in terms of yield and elongation speed (see detailed results). Figure 12 PSUU-2G1K demonstrated excellent amplification capabilities.

[0176]

Claims

1. A DNA polymerase mutant, characterized in that, The amino acid sequence of the DNA polymerase mutant is shown in SEQ ID NO.

1.

2. A method for preparing a DNA polymerase mutant, characterized in that, Includes the following steps: (1) Construction of DNA polymerase mutants: The Pfu DNA polymerase gene was fused with the Sso7d gene (Pfu-Sso7d), and the resulting recombinant plasmid was constructed into the pET-26b(+) vector. A 6×His tag was added to the C-terminus of the fusion protein. The amino acid and nucleotide sequences are shown in SEQ ID NO.1 and SEQ ID NO.

2. (2) Gene cloning and expression: The DNA polymerase mutant recombinant plasmid constructed in step (1) was transformed into Escherichia coli BL21(DE3) cells to induce protein expression and obtain DNA polymerase mutant bacterial culture. (3) Protein purification: The DNA polymerase mutant bacterial culture obtained in step (2) was subjected to heat treatment, precipitation, resuspension and dissolution of the precipitate, affinity chromatography purification, and gradient elution to obtain the DNA polymerase mutant.

3. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the DNA polymerase mutant as described in claim 1, and the nucleotide sequence of the DNA polymerase mutant is shown in SEQ ID NO.

2.

4. A recombinant vector, characterized in that, The recombinant vector contains the gene encoding the DNA polymerase mutant of claim 1.

5. The recombinant vector as described in claim 4, characterized in that, The recombinant vector used is pET-26b(+) as the expression vector.

6. The application of the DNA polymerase mutant as described in claim 1 in the preparation of nucleic acid amplification products.

7. The use of the DNA polymerase mutant as described in claim 1 in the preparation of reagents and / or kits for PCR amplification.

8. The use of the DNA polymerase mutant as described in claim 1 in PCR amplification of oral swab samples or samples containing oral swabs.

9. The use of the DNA polymerase mutant as described in claim 1 in PCR amplification of whole blood samples or samples containing whole blood.

10. The use of the DNA polymerase mutant as described in claim 1 in PCR amplification of bacterial culture samples or samples containing bacterial culture.

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