Recombinant DNA polymerase for sequencing

Abstract

The application provides a recombinant DNA polymerase for sequencing, which is a mutant of 9N DNA polymerase, can recognize 3-O-azido-modified reversible terminators, and maintains high incorporation activity, and thus can be used for second-generation sequencing. The application adopts genetically engineered Escherichia coli to express the mutant of 9N DNA polymerase, and the expressed mutant enzyme realizes soluble expression in the Escherichia coli system and has high catalytic activity.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and more specifically, this invention relates to a recombinant DNA polymerase for sequencing. Background Technology

[0002] DNA polymerases are used in many reactions involving nucleic acid replication, one important area being DNA sequencing. Currently, the mainstream DNA sequencing method is sequencing-by-synthesis (SBS), which relies on the incorporation and detection of reversible terminators by DNA polymerases to achieve large-scale parallel sequencing of DNA molecules, reducing sequencing costs. Unlike natural nucleotides, the 3-OH group of reversible terminators is modified by a larger reversible group, such as 3-O-allyl, 3-O-(2-nitrobenzyl), or 3-O-azido, and also carries a fluorescent group for easy detection. Due to the differences between the two substrates, natural DNA polymerases often have low incorporation efficiency for reversible terminators, or even fail to recognize them. Furthermore, the inherent fidelity of DNA polymerases leads to bias in the incorporation of modified nucleotides, easily causing phasing in the sequencing reaction and limiting its sensitivity. On the other hand, sequencing while synthesizing only extends one base at a time. Subsequently, the reversible terminator 3-OH structure is restored and the fluorescent group is removed through a chemical reaction. However, the linker that connects the fluorescent group still exists on the template DNA. As the sequencing reaction proceeds, the structure of the template DNA and the natural DNA structure become increasingly different, leading to incompatibility between the enzyme and the DNA template. Therefore, it is necessary to modify a new type of DNA polymerase to overcome this limitation.

[0003] 9N° DNA polymerase is a thermostable DNA polymerase derived from *Thermococcus* sp. 9N-7. It was named 9N° DNA polymerase because this bacterium was discovered in a submarine crater in the Pacific Ocean at 9 degrees North latitude. Belonging to the DNA polymerase B family, 9N° DNA polymerase has a molecular weight of approximately 95 kDa. Like most DNA polymerases, it requires magnesium ions as a cofactor to exert its enzymatic activity and also possesses 3'-5' exonuclease activity. Its active site resembles the right-handed conformation of the *E. coli* Klenow fragment, and can be further divided into palm, thumb, and finger domains. Incidental research revealed that 9N° DNA polymerase from *Thermococcus* can effectively recognize and bind reversible terminators and accept a wide range of modified nucleotides as substrates. Therefore, B-family 9N° DNA polymerases are the enzymes of choice for important biotechnologies such as NGS sequencing.

[0004] Therefore, those skilled in the art are dedicated to modifying and recombinantly expressing 9N° DNA polymerase in order to prepare a low-cost 9N° DNA polymerase suitable for industrial production and capable of maintaining high reaction efficiency for use in optical sequencing. Summary of the Invention

[0005] The purpose of this invention is to provide a recombinant DNA polymerase for sequencing, wherein the recombinant DNA polymerase is a 9N° DNA polymerase mutant.

[0006] In a first aspect of the invention, a 9N° DNA polymerase mutant is provided, the 9N° DNA polymerase mutant being mutated at one or more sites selected from the group consisting of: position 141, position 143, position 381, position 485 and position 497, wherein the amino acid residues are numbered as shown in SEQ ID NO.2.

[0007] In another preferred embodiment, the amino selected sequence of the 9N° DNA polymerase mutant has at least 80% homology with SEQ ID NO.2; more preferably, at least 90% homology; most preferably, at least 95% homology; such as at least 96%, 97%, 98%, or 99% homology.

[0008] In another preferred embodiment, the number of mutation sites in the 9N° DNA polymerase mutant is 1-5, preferably 4 or 5.

[0009] In another preferred embodiment, the 9N° DNA polymerase mutant is mutated at amino acid residues 141, 143, 381, 485, and 497 of the 9N° DNA polymerase shown in SEQ ID NO.2; and the amino acid residue at position 141 is mutated to A, the amino acid residue at position 143 is mutated to A, the amino acid residue at position 381 is mutated to K, the amino acid residue at position 485 is mutated to L, and the amino acid residue at position 497 is mutated to V.

[0010] In another preferred embodiment, the amino acid sequence of the 9N° DNA polymerase mutant is shown in SEQ ID NO.4.

[0011] In a second aspect, the present invention provides a polynucleotide molecule that encodes the 9N° DNA polymerase mutant described in the first aspect of the present invention.

[0012] In a third aspect, the present invention provides a carrier containing the nucleic acid molecule described in the second aspect of the present invention.

[0013] In a fourth aspect, the present invention provides a host cell containing a vector or chromosome according to the first aspect of the present invention integrated with nucleic acid molecules according to the second aspect of the present invention.

[0014] In another preferred embodiment, the host cell is a prokaryotic cell or a eukaryotic cell.

[0015] In another preferred embodiment, the prokaryotic cell is Escherichia coli.

[0016] In another preferred embodiment, the eukaryotic cell is a yeast cell.

[0017] A fifth aspect of the present invention provides a method for preparing the 9N° DNA polymerase mutant described in the first aspect of the present invention, comprising the steps of:

[0018] (i) Under suitable conditions, the host cells described in the fourth aspect of the invention are cultured to express the 9N° DNA polymerase mutant; and

[0019] (ii) Isolate the 9N° DNA polymerase mutant.

[0020] In another preferred embodiment, the temperature at which the host cells are cultured in step (i) is 20°C-40°C; preferably 25°C-37°C, such as 35°C.

[0021] In another preferred embodiment, the host cell in step (i) is an Escherichia coli cell.

[0022] In a sixth aspect, the present invention provides a kit comprising the 9N° DNA polymerase mutant described in the first aspect of the present invention.

[0023] A seventh aspect of the present invention provides the use of the 9N° DNA polymerase mutant described in the first aspect of the present invention in the preparation of a gene sequencing kit.

[0024] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0025] Figure 1 Results of 9N° DNA polymerase expression assay;

[0026] Figure 2 This is a standard curve for enzyme activity detection.

[0027] Figure 3 Electrophoresis image for substrate compatibility testing of commercial enzymes;

[0028] Figure 4 This is an electrophoresis diagram of the preferred mutant enzyme substrate compatibility test of the present invention. Detailed Implementation

[0029] Through extensive and in-depth research, the inventors of this invention employed a recombinant fusion 9N° DNA polymerase mutant from genetically engineered *E. coli*. The expressed 9N° DNA polymerase mutant achieved soluble expression in the *E. coli* system with high protein yield and high catalytic activity. It boasts advantages such as short production cycle, easy purification of the expression product, and low cost, enabling the industrial production of the 9N° DNA polymerase mutant. Furthermore, this mutant enzyme can recognize reversible terminators modified with 3-O-azide groups and maintains high incorporation activity, making it applicable to next-generation sequencing. Based on these findings, this invention was completed.

[0030] Before describing this invention, it should be understood that the invention is not limited to the specific methods and experimental conditions described, as such methods and conditions can be varied. It should also be understood that the terminology used herein is intended only to describe particular embodiments and is not intended to be limiting; the scope of the invention will be limited only by the appended claims.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. As used herein, when referring to a specifically enumerated numerical value, the term “about” means that the value can vary from the enumerated value by no more than 1%. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0032] While any methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of this invention, preferred methods and materials are exemplified herein.

[0033] The 9N° DNA polymerase mutant has been commercialized (named Therminator). TM This DNA polymerase carries three mutations: D141A, E143A, and A485L. The D141A and E143A mutations are located in the 3'-5' exonuclease domain, causing it to lose its proofreading activity. The A485 amino acid is located in the finger domain, facing away from the polymerase active site, and does not directly contact the substrate nucleotide. Therefore, this mutation may reduce its ability to distinguish modified nucleotides. However, the optimal substrates for this enzyme are ddNTPs and acyclic nucleotides (AcyNTPs). Another mutant of this enzyme, in addition to the above three mutations, carries the Y409A mutation, which allows it to recognize and bind reversible terminators. However, this polymerase mutant has a lower incorporation efficiency for 3'-terminal modified reversible terminators, requiring longer incubation times and high concentrations of 3'-terminal modified nucleotide reversible terminators to complete the reaction.

[0034] In a preferred embodiment of the present invention, the base sequence of wild-type 9N° DNA polymerase (optimized according to Escherichia coli synonymous codon preference, SEQ ID NO.1):

[0035] ATGATCCTGGACACTGACTACATCACCGAAAATGGCAAACCGGTTATCCGTGTATTCAAAAAGGAAA

[0036] ACGGCGAATTCAAAATCGAATACGATCGCACGTTCGAACCGTACTTTTATGCGCTGCTGAAAGACGA

[0037] CTCCGCGATTGAAGATGTTAAAAAAGTGACGGCGAAGCGTCACGGTACTGTCGTCAAAGTGAAACGT

[0038] GCCGAAAAAGTTCAGAAGAAATTCCTGGGCCGTCCGATCGAAGTATGGAAACTGTACTTCAATCATC

[0039] CGCAGGACGTTCCGGCAATCCGTGATCGCATCCGTGCACACCCTGCTGTAGTGGATATCTACGAATA

[0040] CGACATCCCATTTGCGAAACGTTACCTGATCGACAAAGGCCTGATCCCGATGGAAGGCGACGAAGAA

[0041] CTGACCATGCTGGCATTCGACATCGAAACCCTGTATCACGAAGGTGAGGAATTCGGTACCGGCCCTA

[0042] TCCTGATGATCAGCTACGCTGACGGTTCCGAAGCGCGTGTTATCACCTGGAAAAAAATCGACCTGCC

[0043] GTATGTTGATGTTGTTTCCACTGAAAAGGAAATGATCAAACGCTTCCTGCGTGTAGTGCGTGAAAAA

[0044] GACCCGGATGTACTGATCACCTACAACGGCGACAACTTTGATTTCGCTTATCTGAAAAAACGCTGTG

[0045] AAGAACTGGGTATTAAATTCACTCTGGGTCGTGATGGCTCCGAACCTAAAATCCAGCGTATGGGTGA

[0046] CCGCTTTGCAGTTGAAGTTAAAGGTCGCATCCATTTCGATCTGTATCCAGTCATCCGCCGCACGATT

[0047] AACCTGCCGACCTACACTCTGGAAGCTGTGTATGAGGCAGTATTTGGCAAACCGAAAGAAAAGGTTT

[0048] ATGCGGAAGAAATCGCTCAGGCTTGGGAATCCGGCGAAGGTCTGGAACGTGTTGCGCGCTATTCCAT

[0049] GGAAGATGCGAAAGTGACTTACGAACTGGGTCGCGAATTTTTCCCGATGGAAGCCCAGCTGTCTCGT

[0050]

[0051] In a preferred embodiment of the present invention, the amino acid sequence (SEQ ID NO.2) of the wild-type 9N° DNA polymerase is as follows:

[0052] MILDDTYITENGKPVIRVFKKENGEFKIEYDRTFEPYFYALLKDDSAIEDVKKVTAKRHGTVVKVKRAEKVQKKFLGRPIEVWKLYFNHPQDVPAIRDRIRAHPAVVDIYEYDIPFAKRYLIDKGLIPMEGDEELTMLAFDIETLYHEGEEFGTGPILMISYADGSEARVITWKKIDLPYVDVVSTEKEMIKR FLRVVREKDPDVLITYNGDNFDFAYLKKRCEELGIKFTLGRDGSEPKIQRMGDRFAVEVKGRIHFDLYPVIRRTINLPTYTLEAVYEAVFGKPKEKVYAEEIAQAWESGEGLERVARYSMEDAKVTYELGREFFPMEAQLSRLIGQSLWDVSRSSTGNLVEWFLLRKAYKRNELAPNKPDERELARRRGGYAGG YVKEPERGLWDNIVYLDFRSLYPSIIITHNVSPDTLNREGCKEYDVAPEVGHKFCKDFPGFIPSLLGDLLEERQKIKRKMKATVDPLEKKLLDYRQRAIKILANSFYGYYGYAKARWYCKECAESVTAWGREYIEMVIRELEEKFGFKVLYADTDGLHATIPGADAETVKKKAKEFLKYINPKLPGLLELEYEG FYVRGFFVTKKKYAVIDEEGKITTRGLEIVRRDWSEIAKETQARVLEAILKHGDVEEAVRIVKEVTEKLSKYEVPPPEKLVIHEQITRDLRDYKATGPHVAVAKRLAARGVKIRPGTVISYIVLKGSGRIGDRAIPADEFDPTKHRYDAEYYIENQVLPAVERILKAFGYRKEDLRYQKTKQVGLGAWLKVKGKK

[0053] Based on the published three-dimensional structure (PDB: 5omv) in the database, the amino acid sites of the 9N° DNA polymerase were modified using computer molecular simulation technology. The relevant mutation sites include:

[0054]

[0055] In a preferred embodiment of the present invention, the sequence of the mutant 9N° DNA polymerase according to the present invention is as follows (SEQ ID NO. 4):

[0056] MILDTDYITENGKPVIRVFKKENGEFKIEYDRTFEPYFYALLKDDSAIEDVKKVTAKRHGTVVKVKRAEKVQKKFLGRPIEVWKLYFNHPQDVPAIRDRIRAHPAVVDIYEYDIPFAKRYLIDKGLIPMEGDEELTMLAFAIATLYHEGEEFGTGPILMISYADGSEARVITWKKIDLPYVDVVSTEKEMIKRFLRVVREKDPDVLITYNGDNFDFAYLKKRCEELGIKFTLGRDGSEPKIQRMGDRFAVEVKGRIHFDLYPVIRRTINLPTYTLEAVYEAVFGKPKEKVYAEEIAQAWESGEGLERVARYSMEDAKVTYELGREFFPMEAQLSRLIGQSLWDVSRSSTGNLVEWFLLRKAYKRNELAPNKPDERELARRKGGYAGGYVKEPERGLWDNIVYLDFRSLYPSIIITHNVSPDTLNREGCKEYDVAPEVGHKFCKDFPGFIPSLLGDLLEERQKIKRKMKATVDPLEKKLLDYRQRLIKILANSFYGYVGYAKARWYCKECAESVTAWGREYIEMVIRELEEKFGFKVLYADTDGLHATIPGADAETVKKKAKEFLKYINPKLPGLLELEYEGFYVRGFFVTKKKYAVIDEEGKITTRGLEIVRRDWSEIAKETQARVLEAILKHGDVEEAVRIVKEVTEKLSKYEVPPEKLVIHEQITRDLRDYKATGPHVAVAKRLAARGVKIRPGTVISYIVLKGSGRIGDRAIPADEFDPTKHRYDAEYYIENQVLPAVERILKAFGYRKEDLRYQKTKQVGLGAWLKVKGKK

[0057] The mutant enzyme gene sequence of the present invention can be obtained by conventional methods used by those skilled in the art, such as complete artificial synthesis or PCR synthesis. A preferred synthesis method is asymmetric PCR. Asymmetric PCR uses unequal amounts of a pair of primers, and PCR amplification produces a large amount of single-stranded DNA (ssDNA). This pair of primers is called the non-restriction primer and the restriction primer, and their ratio is generally 50-100:1. In the first 10-15 cycles of the PCR reaction, the amplification product is mainly double-stranded DNA, but when the restriction primer (low concentration primer) is consumed, PCR guided by the non-restriction primer (high concentration primer) will produce a large amount of single-stranded DNA. The primers used for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein, and can be synthesized using conventional methods. The amplified DNA / RNA fragments can be separated and purified using conventional methods such as gel electrophoresis.

[0058] The mutant enzyme of the present invention can be expressed or produced using conventional recombinant DNA technology, including the following steps:

[0059] (1) Transform or transduce suitable host cells using a polynucleotide encoding the protein of the present invention, or using a recombinant expression vector containing the polynucleotide;

[0060] (2) Culture the host cells in a suitable culture medium;

[0061] (3) Isolate and purify the target protein from the culture medium or cells to obtain the target enzyme.

[0062] Methods well known to those skilled in the art can be used to construct expression vectors containing the encoding DNA sequence of the enzyme of the present invention and suitable transcription / translation control signals, preferably commercially available vectors such as pET28. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. The DNA sequence can be efficiently ligated to an appropriate promoter in the expression vector to guide mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. Furthermore, the expression vector preferably contains one or more selective marker genes to provide phenotypic traits for selecting transformed host cells.

[0063] The recombinant vector includes, in the 5' to 3' direction: a promoter, a target gene, and a terminator. If desired, the recombinant vector may also include the following elements: a protein purification tag; a 3' polynucleotide signal; a non-translated nucleic acid sequence; a transport and targeting nucleic acid sequence; a selection marker (antibiotic resistance gene, fluorescent protein, etc.); an enhancer; or an operator.

[0064] The methods used to prepare recombinant vectors are well known to those skilled in the art. Expression vectors can be bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses, or other vectors. In short, any plasmid and vector can be used as long as it can replicate and remain stable within the host.

[0065] Those skilled in the art can construct vectors containing the promoter and / or target gene sequence of this invention using well-known methods. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc.

[0066] The expression vector of this invention can be used to transform suitable host cells to enable the host to transcribe target RNA or express target protein. Host cells can be prokaryotic cells, such as *Escherichia coli*, *Corynebacterium glutamicum*, *Brevibacterium flavum*, *Streptomyces*, and *Agrobacterium*; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as plant cells. Those skilled in the art will understand how to select appropriate vectors and host cells. Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote (such as *E. coli*), it can be treated with CaCl2 or electroporation. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate co-precipitation, conventional mechanical methods (such as microinjection, electroporation, liposome packaging, etc.). Transformation of plants can also be performed using methods such as *Agrobacterium* transformation or gene gun transformation, for example, leaf disc transformation, embryo transformation, and flower bud soaking. Transformed plant cells, tissues, or organs can be regenerated into plants using conventional methods to obtain transgenic plants.

[0067] The term "operable link" refers to linking a target gene intended for transcription and expression to its control sequence in a manner conventional in the art for expression.

[0068] Cultivation of engineered bacteria and fermentation production of target protein

[0069] After obtaining engineered cells, they can be cultured under suitable conditions to express the protein encoded by the gene sequence of this invention. Depending on the host cell, the culture medium used can be selected from various conventional media, and cultured under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature change or chemical induction), and the cells are cultured for a further period.

[0070] In this invention, conventional fermentation conditions can be used. Representative conditions include (but are not limited to):

[0071] (a) In terms of temperature, the fermentation and induction temperature of enzymes is maintained at 25-37°C;

[0072] (b) Regarding the pH value during the induction period, the pH during the induction period should be controlled between 3 and 9;

[0073] (c) Regarding dissolved oxygen (DO), DO is controlled at 10-90%, and the maintenance of dissolved oxygen can be achieved by introducing an oxygen / air mixture.

[0074] (d) In terms of feeding, the types of feeding should include carbon sources such as glycerol, methanol, and glucose, which can be fed alone or in combination;

[0075] (e) Regarding the IPTG concentration during the induction period, conventional induction concentrations can be used in this invention, typically with the IPTG concentration controlled between 0.1 and 1.5 mM;

[0076] (f) There are no particular restrictions on the induction time, which is usually 2-20 hours, preferably 5-15 hours.

[0077] The target protein of this invention is located intracellularly in *E. coli* cells. Host cells are collected using a centrifuge, and then the host cells are broken down using high pressure, mechanical force, enzymatic hydrolysis, or other cell disruption methods to release the recombinant protein. High pressure is preferred. The host cell lysate can be initially purified by methods such as flocculation, salting out, and ultrafiltration before further purification by chromatography or ultrafiltration, or it can be directly purified by chromatography.

[0078] Chromatographic techniques include cation exchange chromatography, anion exchange chromatography, gel filtration chromatography, hydrophobic chromatography, affinity chromatography, and other techniques. Commonly used chromatographic methods include:

[0079] 1. Anion exchange chromatography:

[0080] Anion exchange chromatography media include (but are not limited to): Q-Sepharose and DEAE-Sepharose. If the salt concentration of the fermentation sample is high, affecting its binding with the ion exchange medium, the salt concentration needs to be reduced before ion exchange chromatography. The sample can be diluted, ultrafiltered, dialyzed, or subjected to gel filtration chromatography to replace the equilibration buffer until it resembles the equilibration buffer system of the corresponding ion exchange column. Then, the sample is loaded and eluted using a gradient of salt concentration or pH.

[0081] 2. Hydrophobic chromatography:

[0082] Hydrophobic chromatography media include (but are not limited to): Phenyl-Sepharose, Butyl-Sepharose, and Octyl-Sepharose. The sample is loaded with salt by adding NaCl, (NH4)2SO4, etc., to increase the salt concentration, and then eluted by decreasing the salt concentration. Hydrophobic chromatography removes contaminating proteins with significantly different hydrophobicities.

[0083] 3. Gel filtration chromatography

[0084] Hydrophobic chromatography media include (but are not limited to): Sephacryl, Superdex, and Sephadex-type media. The buffer system can be changed during gel filtration chromatography, or further purification can be performed.

[0085] 4. Affinity Chromatography

[0086] Affinity chromatography media include (but are not limited to): HiTrap TM HeparinHPColumns.

[0087] 5. Membrane filtration

[0088] Ultrafiltration media include: organic membranes such as polysulfone membranes, inorganic membranes such as ceramic membranes, and metal membranes. Membrane filtration can achieve purification and concentration.

[0089] The main advantages of this invention are:

[0090] (1) The 9N° DNA polymerase mutant of the present invention can recognize the reversible terminator modified by 3-O-azido group and maintain high incorporation activity, and can be applied to optical sequencing.

[0091] (2) The 9N° DNA polymerase mutant of the present invention can achieve large-scale soluble expression in the Escherichia coli expression system, is easy to purify with high yield, and maintains high biocatalytic activity.

[0092] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions as described in *Molecular Cloning: A Laboratory Manual* by Sambrook J. et al. (translated by Huang Peitang et al., Beijing: Science Press, 2002), or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated. Unless otherwise specified, all experimental materials and reagents used in the following embodiments are commercially available.

[0093] Example 1: Construction, expression, and purification of Escherichia coli 9N° DNA polymerase plasmid

[0094] 1) Based on the protein sequence of wild-type 9N° DNA polymerase (SEQ ID NO.2) and in accordance with the experimental design requirements of this invention, the coding base sequences of wild-type and different mutants were obtained after optimization by E. coli synonymous codon preference. Each base sequence was ligated into the vector pET-28a(+) and fused with the C-terminus expression (His)6 tag. The sequences were synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.

[0095] Table 1

[0096] 1 wild type 2 D141A, E143A, A485L, L408A, P410G 3 D141A, E143A, A485L, E578G, E580A 4 D141A, E143A, A485L, V282F, K464L 5 D141A, E143A, A485L, T267G, Q483K 6 D141A, E143A, A485L 7 D141A, E143A, A485L, N491V, Y494H 8 D141A, E143A, R381K, A485L, Y497V

[0097] 2) Recombinant plasmid was transformed into Escherichia coli BL21(DE3)

[0098] Take 1 μL of plasmid and add it to 30 μL of competent E. coli BL21(DE3) cells under ice bath conditions. Incubate on ice for 20 min, then heat shock at 42°C for 45 s, immediately place on ice for 2 min, add 400 μL of antibiotic-free SOC medium, and incubate at 37°C with shaking at 220 rpm for 50 min. Spread 100 μL of the bacterial culture evenly onto LB agar plates containing 100 μg / mL kanamycin and incubate overnight at 37°C.

[0099] 3) Expression of the target protein

[0100] Select single clones from step 2) and aseptically inoculate them into SOC medium containing 100 μg / mL kanamycin resistance. Incubate at 37°C with shaking at 220 rpm until OD reaches 100%. 600 IPTG was used for induction at a concentration between 0.6 and 0.8 (final concentration 0.1 mM), and the mixture was incubated overnight at 18°C ​​with shaking. The control group (without IPTG) was used. The mixture was then incubated at 37°C for 3 hours. Each experiment was repeated once. Samples were taken and sonicated for SDS-PAGE analysis. The results showed that 9N° DNA polymerase could be expressed solublely in the supernatant in the culture medium of strains 4, 5, 6, 7, and 8. The yield of soluble target protein in strain 8 was higher than that in other strains. No soluble target protein was detected in strains 2 and 3. The molecular weight of the target protein was approximately 100 kDa, which is consistent with the protein size predicted by the Expasy website (95 kDa). Figure 1 ).

[0101] 4) Purification of 9N° DNA polymerase

[0102] The solution formulation used to prepare the sample is as follows:

[0103] Buffer A: 50mM Tris, 50mM NaCl, 5% Glycerol, pH 8.0

[0104] Buffer B: 50mM Tris, 50mM NaCl, 500mM Imidazole, 5% Glycerol, pH 8.0

[0105] Buffer C: 100mM Tris, 1M NaCl, 10% Glycerol, pH 8.0

[0106] Lysis Buffer: 50mM Tris, 300mM NaCl, 5% Glycerol, pH 8.0

[0107] 9N dialysis buffer: 10 mM Tris-HCl, 100 mM KCl, 1 mM DTT, 0.1 mM EDTA, 50% Glycerol, pH 7.4

[0108] The specific operating steps are as follows:

[0109] 1.5 L of bacterial culture was cultured in a shake flask using SOC medium, with expression conditions consistent with those for the target protein in step 3). The bacterial cells were collected by centrifugation. Approximately 20 g of bacterial cells were weighed and resuspended in 100 ml of Lysis Buffer on ice. Cell disruption was performed by sonication: Ф10 probe, 20% power, 5.5 s on, 9.9 s off, for 30 min. After disruption, the sample was heat-treated in a 70℃ water bath for 20 min. The sample was centrifuged at 20000 rpm at 4℃ for 35 min, and the supernatant was collected and filtered through a 0.22 μm membrane. The supernatant was subjected to Ni-column affinity chromatography using a HisTrap column. TM Linear elution was performed using HP, 0-60% Buffer B. The eluent containing the main peak was then subjected to ion exchange elution. A HisTrap purification column was used. TM SP-HP was linearly eluted with 0-60% Buffer C to obtain 90 ml of the target protein solution.

[0110] The purified sample was dialyzed overnight in dialysate, and the protein concentration was determined by SDS-PAGE grayscale analysis.

[0111] The protein concentrations of the purified samples are shown in Table 2 below.

[0112] Table 2

[0113]

[0114]

[0115] The results showed that the protein concentration and protein yield of the purified sample were the highest.

[0116] Example 2: Assay of Recombinant 9N° DNA Polymerase Activity

[0117] 1. Experimental Objective

[0118] Indication of 9N° DNA polymerase activity.

[0119] 2. Experimental Materials

[0120] Sample: 9N° DNA polymerase

[0121] Equipment and reagents: Real-time quantitative PCR system, Picogreen, λDNA (Merck).

[0122] Substrate T2 (SEQ ID NO.5):

[0123] The above substrate was synthesized by 5′tagcgaatgatgtgcacctaatcgaTGCTCCCGCGGCCGatcttagCGGCCGCGGGAGCA3′, purified by HPLC, and prepared as a 100 pmol / μL stock solution according to experimental requirements.

[0124] 4) Preparation of 9N° enzyme dilution solution:

[0125] 10mM Tris-HCl, 100mM KCl, 1mM DTT, 0.1mM EDTA, 50% Glycerol, (pH 7.4@25℃)

[0126] 3. Experimental Procedure

[0127] 3.1 Prepare 10×PCR Buffer according to the following table:

[0128] <![CDATA[1M(NH4)2SO4]]> 0.5ml 1M KCl 5ml 10% Triton X-100 1ml <![CDATA[ddH2O]]> 1ml

[0129] 3.2 Prepare the λDNA pre-staining solution according to the table below:

[0130] picogreen 15ul <![CDATA[ddH2O]]> 255ul

[0131] 3.3λDNA was diluted in a gradient according to the table below:

[0132]

[0133]

[0134] 3.4 Dilution of the test sample:

[0135] 9N° enzyme samples are diluted with 9N° enzyme dilution buffer. Dilution factors are 1 / 100, 1 / 200, 1 / 400, 1 / 800, 1 / 1600, and 1 / 3200. The dilution factor can be adjusted according to actual conditions to achieve the same protein concentration.

[0136] 3.5 Reaction System Configuration (Single Reaction System)

[0137] <![CDATA[25mM MgCl2]]> 2ul T2(100p) 0.1ul 25mM dNTP 0.2ul picogreen 0.5ul water 13.9ul polymerase sample to be tested 1.3ul

[0138] For each dilution gradient, λDNA and λDNA pre-staining solution were mixed thoroughly at a 1:1 ratio. The 0 μg / ml λDNA group served as a control. After calculating the required number of wells (N), the components of the above reaction system, except for the 9N° enzyme, were mixed thoroughly in N+3 increments, and 18.7 μL was spotted into each well of a 96-well plate. For each λDNA concentration gradient, 20 μL was spotted into each well of the 96-well plate. Each concentration gradient of sample enzyme and λDNA was performed in at least 3 replicates. Then, 1.3 μL of diluted 9N° enzyme was added to each well (operated on ice), vortexed, and centrifuged. 1.3 μL of 9N° enzyme dilution was added to the NTC group. No sample enzyme was added to the λDNA group. The 96-well plate was placed in a qPCR instrument, and SYBR reads were performed at (74℃ 14s, 74℃ 2s) × 120 cycles, 74℃ 2s, with real-time monitoring of fluorescence values.

[0139] 4. Experimental Results:

[0140] 4.1 Construction of the λDNA Standard Curve

[0141] 1) Export the Q5 test data to an Excel spreadsheet. Select the fluorescence values ​​of the λDNA standard and the λDNA NTC group at the 30th cycle. Calculate the average of the three data sets at each λDNA concentration. Subtract the average of the three data sets in the NTC group to obtain the net fluorescence value. Plot a linear standard curve with the amount of λDNA added on the x-axis and the net fluorescence value on the y-axis, and set its R-value to... 2 >0.99, standard curve as follows Figure 2 As shown:

[0142] Export the Q5 test data to an Excel spreadsheet. Select the fluorescence values ​​at the 30th cycle of each gradient 9N° enzyme experimental group and the control NTC group. Calculate the average of the three data points for each gradient 9N° enzyme experimental group, and subtract the average of the three data points for the NTC group to obtain the net fluorescence value. Substitute the net fluorescence value into the λDNA standard curve to calculate the amount of DNA generated, A1. Divide A1 / 649 to obtain the amount of dNTPs consumed in the reaction, A2, in nmol. Finally, calculate the 9N° enzyme activity using the formula A2 * 0.449 * dilution factor.

[0143] Enzyme activity is defined as the amount of enzyme required to incorporate 10 nmol dNTPs at 74℃ for 30 min, which is defined as 1 U.

[0144] The measurement results are shown in Table 3 below.

[0145] Table 3

[0146] 1 8.5 7.2 11.6 9.1 4 0 0 0 0.0 5 18.5 24.3 22.6 21.8 6 54.9 53.6 49.8 52.8 7 10.6 12.5 13.2 12.1 8 105.6 109.5 106.3 107.1 Commercial enzymes 52.3 58.7 55.1 55.4

[0147] Note: The product enzyme is Therminator. TMDNA polymerase (NEB).

[0148] Results: λDNA concentration standard curve R 2 >0.99, strains 8 and 6 showed high activity, approximately 107 U / μL and 53 U / μL, respectively. Based on inter-batch enzyme assay results, when the CV value is below 10% and the amount of dNTPs consumed is between 0.01 and 0.14 nmol, 9N ° The enzyme activity results across different gradient ranges were similar. The enzyme activity of this invention is significantly higher than that of the commercial enzyme (Therminator DNA polymerase).

[0149] Example 3: Recombinant 9N° DNA Polymerase Substrate Compatibility Test

[0150] 1. Experimental Objective

[0151] Testing the performance of 9N° DNA polymerase

[0152] 2. Experimental Materials

[0153] 1) Sample: 10X Reaction Buffer (NEB), each 9N° DNA polymerase mutant.

[0154] 2) Equipment: Chemiluminescence fluorescence imaging system, gene analyzer

[0155] 3) Substrate:

[0156] 9N Test-A (SEQ ID NO.6):

[0157] CGATCACGATCACGATCACGATCACGATCACGATCACCGCTGATGTGCATGCTGTTGTTTTTTTACAACAGCATGCACATCAGCGTG

[0158] 9N Test-T (SEQ ID NO.7):

[0159] CGATCACGATCACGATCACGATCACGATCACGATCACCGCTGATGTGCATGCTGTTGTTTTTTTACAACAGCATGCACATCAGCG

[0160] 9N Test-G (SEQ ID NO.8):

[0161] CGATCACGATCACGATCACGATCACGATCACGATCACCGCTGATGTGCATGCTGTTGTTTTTTTACAACAGCATGCACATCAGC

[0162] 9N Test-C (SEQ ID NO.9):

[0163] CGATCACGATCACGATCACGATCACGATCACGATCACCGCTGATGTGCATGCTGTTGTTTTTTTACAACAGCATGCACATCAG

[0164] The sequencing substrates consist of four nucleotides: A, T, G, and C. Each nucleotide has its 3' OH group replaced by an azide group and carries a fluorescent group. Base A fluoresces in both red and green channels, base T fluoresces in the green channel, base C fluoresces in the red channel, and base G does not fluoresce in either red or green channels. These four templates were synthesized externally (HPLC purified, purity >90%), and sterile water was added to a final concentration of 100 pmol / μL. The solution was then diluted 10-fold with sterile water to prepare a working solution with a concentration of 10 pmol / μL.

[0165] 4) Experimental materials

[0166] 10X sequencing substrate, GeneScan 600LIZ Size Standard v2.0, HiDi-Formamide (deionized formamide), 6X DNA Loading Buffer.

[0167] 3. Experimental Procedure

[0168] 3.1 Substrate compatibility test

[0169] 1) Configuration of the reaction buffer

[0170] 10X sequencing substrate, 10X Prepare a substrate concentration of 1.1X Mix by mixing Reaction Buffer and deionized water in a 1:9 ratio. Mix 9N° enzyme and 1.1X Mix in a 1:9 ratio to obtain an enzyme concentration of 5 U / μL.

[0171] 2) Reaction system configuration (single-person reaction):

[0172]

[0173]

[0174] Calculate the required number of wells (N), and use N+3 wells. Add 9 μL of the mixture to each well in an 8-row configuration, followed by 1 μL of annealed 9N°Test-A / T / G / C per well. Vortex and centrifuge. Place in a PCR instrument and react at 60°C for 4 min and 95°C for 10 min. Add 2 μL of 6X DNA Loading Buffer (dye-free) to the reaction product, bringing the final concentration to 0.1% SDS. Perform 15% Urea-PAGE electrophoresis, and scan the gel using a chemiluminescence fluorescence imaging system for red and green light channels.

[0175] 4 Experimental Results

[0176] Test results showed that enzyme samples from strains 1, 5, and 6, as well as commercial enzyme samples, were incompatible with the sequencing substrate, with no single-base extension band observed at 100 bp. Strain 7 enzyme sample showed a very weak single-base extension band signal at 100 bp, indicating that this mutant had low incorporation efficiency. Representative electrophoresis images of commercial enzyme substrate compatibility tests are shown below. Figure 3 As shown in the figure, no single-base extension band at 100bp was observed in either red or green light imaging, indicating that the commercial enzyme sample is incompatible with sequencing substrates.

[0177] Electrophoresis diagram of substrate compatibility test of 9N° DNA polymerase for strain 8 is shown below. Figure 4 As shown in the image, red light scanning reveals template groups A and C, with a clear single-base extension band at 100 bp; green light scanning reveals template groups A and T, with a clear single-base extension band at 100 bp. This demonstrates that the 9N° DNA polymerase mutant is compatible with sequencing substrates.

[0178] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A 9N 。 DNA polymerase mutant, characterized by... The 9N 。 The amino acid sequence of the DNA polymerase mutant is shown in SEQ ID NO.

4.

2. A polynucleotide molecule, characterized in that, The polynucleotide molecule encodes the 9N of claim 1. 。 DNA polymerase mutant.

3. A carrier, characterized in that, The carrier contains the polynucleotide molecule as described in claim 2.

4. A host cell, characterized in that, The host cell contains the vector of claim 3 or the chromosome is integrated with the polynucleotide molecule of claim 2.

5. The host cell as described in claim 4, characterized in that, The host cell is a prokaryotic cell or a eukaryotic cell.

6. The host cell as described in claim 5, characterized in that, The prokaryotic cells are Escherichia coli cells.

7. A method for preparing the 9N according to claim 1 。 The method for DNA polymerase mutants, characterized in that... Including the following steps: (i) Under suitable conditions, the host cells of claim 4 are cultured to express the 9N. 。 DNA polymerase mutants; and (ii) Separate the 9N 。 DNA polymerase mutant.

8. The method as described in claim 7, characterized in that, The host cell in step (i) is an Escherichia coli cell.

9. A reagent kit, characterized in that, The kit comprises the 9N as described in claim 1. 。 DNA polymerase mutant.

10. The 9N as described in claim 1 。 Application of DNA polymerase mutants in the preparation of gene sequencing kits.

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