DNA polymerase mutant for improving long fragment amplification efficiency and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a DNA polymerase mutant for improving long fragment amplification efficiency and application of the DNA polymerase mutant. According to the invention, the DNA polymerase mutants KODL and KODW with enhanced continuous synthesis capability are successfully constructed by carrying out combined mutation of specific amino acid sites on the KOD DNA polymerase, the amplification efficiency of the mutants on a long-fragment DNA template (such as 30kb) is remarkably improved on the basis of retaining the amplification efficiency on fragments with conventional lengths, and the application of the mutants to the preparation of the long-fragment DNA template is facilitated. The technical bottleneck of the existing variable temperature amplification enzyme in long fragment amplification is broken through, and an efficient and reliable enzymology tool is provided for molecular biology application of long fragment gene cloning, whole genome amplification, complex structure analysis and the like.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a DNA polymerase mutant that improves the amplification efficiency of long fragments and its applications. Background Technology

[0002] DNA polymerases are responsible for genome replication and maintenance, a crucial function for the accurate transmission of genetic information from generation to generation. DNA polymerases are broadly classified into two categories: isothermal amplification DNA polymerases (such as KOD DNA polymerase) and isothermal amplification DNA polymerases (such as Phi29 DNA polymerase). Isothermal amplification DNA polymerases, relying on their strong strand substitution ability, can amplify circular templates to obtain long fragment products. For example, Phi29 DNA polymerase can obtain approximately 70kb of single-stranded amplification products using circular templates. However, due to this characteristic, it cannot be used for gene amplification and product accumulation of conventional linear double-stranded DNA fragments. Isothermal amplification DNA polymerases overcome this limitation. Although they lack strand substitution ability, their amplification process requires temperature-controlled melting followed by primer recombination for DNA amplification, allowing for the continuous accumulation of double-stranded DNA products. However, the persistence of isothermal amplification DNA polymerases is not as good as that of isothermal amplification DNA polymerases. Therefore, improving the persistence of isothermal DNA polymerases and their ability to amplify long double-stranded DNA fragments is crucial for expanding their applications. Summary of the Invention

[0003] The first aspect of the present invention is to provide a DNA polymerase mutant.

[0004] A second aspect of the present invention is to provide a fusion protein.

[0005] A third aspect of the present invention is to provide a nucleic acid molecule.

[0006] A fourth aspect of the present invention is to provide a carrier.

[0007] The fifth aspect of this invention aims to provide a host cell.

[0008] The sixth aspect of this invention is to provide a reagent kit.

[0009] The seventh aspect of this invention aims to provide a preparation method.

[0010] The object of the eighth aspect of the present invention is to provide an application.

[0011] The ninth aspect of this invention aims to provide a method for amplifying long DNA templates.

[0012] To achieve the above-mentioned objectives of this invention, the technical solution adopted by this invention is as follows: In a first aspect, the present invention provides a DNA polymerase mutant, the amino acid sequence of which is shown in SEQ ID NO: 3 or SEQ ID NO: 5.

[0013] In some embodiments of the present invention, the DNA polymerase mutants include KODL and KODW.

[0014] Among them, KODL has D141A, E143A, N210D, and Y311F mutations compared to KODWT; KODW has D141A, I142R, E143A, N210D, and Y311F mutations compared to KODWT.

[0015] A second aspect of the present invention provides a fusion protein comprising the DNA polymerase mutant described in the first aspect of the present invention and a tag that assists in purification or expression.

[0016] In some embodiments of the present invention, the tag includes at least one of His, Flag, HA, GST, GFP, and Biotin.

[0017] In some embodiments of the present invention, the tag is located at the N-terminus and / or C-terminus of the DNA polymerase mutant.

[0018] A third aspect of the present invention provides a nucleic acid molecule that expresses the DNA polymerase mutant described in the first aspect of the present invention or the fusion protein described in the second aspect of the present invention.

[0019] In some embodiments of the present invention, the nucleic acid molecule includes a sequence as shown in SEQ ID NO: 4 or SEQ ID NO: 6.

[0020] A fourth aspect of the present invention provides a carrier comprising the nucleic acid molecule described in the third aspect of the present invention.

[0021] In some embodiments of the present invention, the carrier includes a eukaryotic cell carrier or a prokaryotic cell carrier.

[0022] A fifth aspect of the present invention provides a host cell comprising the nucleic acid molecule described in the third aspect of the present invention or the vector described in the fourth aspect of the present invention.

[0023] In some embodiments of the present invention, the host cell includes a eukaryotic cell or a prokaryotic cell.

[0024] In some embodiments of the present invention, the eukaryotic cells include yeast cells.

[0025] In some embodiments of the present invention, the prokaryotic cells include Escherichia coli.

[0026] A sixth aspect of the present invention provides a kit comprising the DNA polymerase mutant described in the first aspect of the present invention or the fusion protein described in the second aspect of the present invention, and reaction aids.

[0027] In some embodiments of the present invention, the reaction aids include conventional materials for DNA amplification such as reaction buffer, metal ions, and dNTPs.

[0028] A seventh aspect of the present invention provides a method for preparing the DNA polymerase mutant of the first aspect of the present invention or the fusion protein of the second aspect of the present invention, comprising the following steps: The host cells described in the fifth aspect of this invention are cultured, and DNA polymerase mutants or fusion proteins are isolated and purified.

[0029] Specifically, the preparation method of DNA polymerase includes the following steps: 1) Provide the gene expression sequence for expressing DNA polymerase; 2) The gene expression sequence was ligated into the gene expression vector pET-21a; 3) The gene expression vector was transformed into host cells BL21 to obtain recombinant cells; 4) Induce expression in recombinant cells and collect bacterial cells; 5) After resuspending, the bacterial cells are disrupted by sonication, and the supernatant is collected; 6) Utilizing Ni 2+ The supernatant was purified using an affinity column, and the purified protein was collected. 7) The protein described in step six was concentrated using an ultrafiltration column to obtain DNA polymerase.

[0030] In some embodiments of the present invention, the temperature for culturing the host cells is 20°C. 40℃; preferably 25℃ 37℃.

[0031] An eighth aspect of the present invention provides the use of the DNA polymerase mutant described in the first aspect of the present invention or the fusion protein described in the second aspect of the present invention in amplifying long DNA templates.

[0032] In some embodiments of the present invention, the long DNA template refers to a DNA template of 10kb or more.

[0033] A ninth aspect of the present invention provides a method for amplifying a long DNA template, comprising the following steps: DNA synthesis is performed using the DNA polymerase mutant described in the first aspect of the present invention, the fusion protein described in the second aspect of the present invention, or the kit described in the sixth aspect of the present invention.

[0034] The beneficial effects of this invention are: This invention successfully constructed DNA polymerase mutants KODL and KODW with enhanced continuous synthesis capabilities by combining specific amino acid sites in KOD DNA polymerase. While retaining the amplification efficiency for conventional length fragments, these mutants significantly improved the amplification efficiency for long DNA templates (such as 30kb), breaking through the technical bottleneck of existing temperature-dependent amplification enzymes in long fragment amplification. This provides an efficient and reliable enzymatic tool for molecular biology applications such as long fragment gene cloning, whole genome amplification, and complex structure analysis. Attached Figure Description

[0035] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 The results show the purification of the DNA polymerase mutant of this invention.

[0036] Figure 2 Results of DNA polymerase activity assay for amplifying a 6kb target band.

[0037] Figure 3 Results of an activity experiment for DNA polymerase to amplify a 30kb target band. Detailed Implementation

[0038] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0039] Example 1 Mutation screening of DNA polymerase In this embodiment, key sites of the KODWT wild-type DNA polymerase were mutated to construct two mutant DNA polymerases, named KODL and KODW.

[0040] KODL has D141A, E143A, N210D, and Y311F mutations compared to KODWT.

[0041] Compared to KODWT, KODW exhibits mutations in D141A, I142R, E143A, N210D, and Y311F.

[0042] Among them, the amino acid sequence of DNA polymerase KODWT is as follows: MILDTDYITEDGKPVIRIFKKENGEFKIEYDRTFEPYFYALLKDDSAIEEVKKITAERHGTVVTVKRVEKVQKKFLGRPVEVWKLYFTHPQDVPAIRDKIREHPAVIDIYEYDIPFAKRYLIDKGLVPMEGDEELKMLAFDIETLYHEGEEFAEGPILMISYADEEGARVITWKNVDLPYVDVVSTEREMIKRFLRVVKEKDPDVLITYNGDNFDFAYLKKRCEKLGINFALGRDGSEPKIQRMGDRFAVEVKGRIHFDLYPVIRRTINLPTYTLEAVYEAVFGQPKEKVYAEEITTAWETGENLERVARYSMEDAKVTYELGKEFLPMEAQLSRLIGQSLWDVSRSSTGNLVEWFLLRKAYERNELAPNKPDEKELARRRQSYEGGYVKEPERGLWENIVYLDFRSLYPSIIITHNVSPDTLNREGCKEYDVAPQVGHRFCKDFPGFIPSLLGDLLEERQKIKKKMKATIDPIERKLLDYRQRAIKILANSYYGYYGYARARWYCKECAESVTAWGREYITMTIKEIEEKYGFKVIYSDTDGFFATIPGADAETVKKKAMEFLKYINAKLPGALELEYEGFYKRGFFVTKKKYAVIDEEGKITTRGLEIVRRDWSEIAKETQARVLEALLKDGDVEKAVRIVKEVTEKLSKYEVPPEKLVIHEQITRDLKDYKATGPHVAVAKRLAARGVKIRPGTVISYIVLKGSGRIGDRAIPFDEFDPTKHKYDAEYYIENQVLPAVERILRAFGYRKEDLRYQKTRQVGLSAWLKPKGTLE (SEQ ID NO: 1).

[0043] The nucleotide sequence of DNA polymerase KODWT is as follows:

[0044] The amino acid sequence of DNA polymerase KODL is as follows: MILDTDYITEDGKPVIRIFKKENGEFKIEYDRTFEPYFYALLKDDSAIEEVKKITAERHGTVVTVKRVEKVQKKFLGRPVEVWKLYFTHPQDVPAIRDKIREHPAVIDIYEYDIPFAKRYLIDKGLVPMEGDEELKMLAF A I A TLYHEGEEFAEGPILMISYADEEGARVITWKNVDLPYVDVVSTEREMIKRFLRVVKEKDPDVLITY D GDNFDFAYLKKRCEKLGINFALGRDGSEPKIQRMGDRFAVEVKGRIHFDLYPVIRRTINLPTYTLEAVYEAVFGQPKEKVYAEEITTAWETGENLERVAR F SMEDAKVTYELGKEFLPMEAQLSRLIGQSLWDVSRSSTGNLVEWFLLRKAYERNELAPNKPDEKELARRRQSYEGGYVKEPERGLWENIVYLDFRSLYPSIIITHNVSPDTLNREGCKEYDVAPQVGHRFCKDFPGFIPSLLGDLLEERQKIKKKMKATIDPIERKLLDYRQRAIKILANSYYGYYGYARARWYCKECAESVTAWGREYITMTIKEIEEKYGFKVIYSDTDGFFATIPGADAETVKKKAMEFLKYINAKLPGALELEYEGFYKRGFFVTKKKYAVIDEEGKITTRGLEIVRRDWSEIAKETQARVLEALLKDGDVEKAVRIVKEVTEKLSKYEVPPEKLVIHEQITRDLKDYKATGPHVAVAKRLAARGVKIRPGTVISYIVLKGSGRIGDRAIPFDEFDPTKHKYDAEYYIENQVLPAVERILRAFGYRKEDLRYQKTRQVGLSAWLKPKGTLE (SEQ ID NO: 3).

[0045] The nucleotide sequence of DNA polymerase KODL is as follows:

[0046] The amino acid sequence of DNA polymerase KODW is as follows: MILDTDYITEDGKPVIRIFKKENGEFKIEYDRTFEPYFYALLKDDSAIEEVKKITAERHGTVVTVKRVEKVQKKFLGRPVEVWKLYFTHPQDVPAIRDKIREHPAVIDIYEYDIPFAKRYLIDKGLVPMEGDEELKMLAF ARA TLYHEGEEFAEGPILMISYADEEGARVITWKNVDLPYVDVVSTEREMIKRFLRVVKEKDPDVLITY D GDNFDFAYLKKRCEKLGINFALGRDGSEPKIQRMGDRFAVEVKGRIHFDLYPVIRRTINLPTYTLEAVYEAVFGQPKEKVYAEEITTAWETGENLERVAR F SMEDAKVTYELGKEFLPMEAQLSRLIGQSLWDVSRSSTGNLVEWFLLRKAYERNELAPNKPDEKELARRRQSYEGGYVKEPERGLWENIVYLDFRSLYPSIIITHNVSPDTLNREGCKEYDVAPQVGHRFCKDFPGFIPSLLGDLLEERQKIKKKMKATIDPIERKLLDYRQRAIKILANSYYGYYGYARARWYCKECAESVTAWGREYITMTIKEIEEKYGFKVIYSDTDGFFATIPGADAETVKKKAMEFLKYINAKLPGALELEYEGFYKRGFFVTKKKYAVIDEEGKITTRGLEIVRRDWSEIAKETQARVLEALLKDGDVEKAVRIVKEVTEKLSKYEVPPEKLVIHEQITRDLKDYKATGPHVAVAKRLAARGVKIRPGTVISYIVLKGSGRIGDRAIPFDEFDPTKHKYDAEYYIENQVLPAVERILRAFGYRKEDLRYQKTRQVGLSAWLKPKGTLE (SEQ ID NO: 5).

[0047] The nucleotide sequence of DNA polymerase KODW is as follows:

[0048] Example 2 Purification and Expression of DNA Polymerase 1. Expression of DNA polymerase The three DNA polymerase gene expression sequences from Example 1 were transformed into *E. coli* competent cells BL21 (Shanghai Tulugang Biotechnology Co., Ltd., catalog number CC96107) (a 6×His tag sequence was added to the 3' end of the gene for easier expression). After resistance selection, 5 μL of the inoculum was inoculated into 5 mL of LB broth and incubated overnight at 37°C with shaking. Then, it was transferred to 200 mL of LB broth and incubated at 37°C for 5 h with shaking. OD 600 When the concentration of the bacterial culture reached 1, the bacterial culture was removed and cooled at 4°C. After cooling, IPTG was added to a final concentration of 0.6 mM for induction, and the culture was continued at 20°C for 24 hours. The fusion polymerase was purified by Ni column affinity chromatography, and the polymerase was concentrated using an AMICON μLTRA 15 mL 50K ultrafiltration column (Millipore, catalog number: UFC905024).

[0049] 2. SDS-PAGE protein electrophoresis was used to verify the size and purity of the fusion protein. The KODWT, KODL, and KODW DNA polymerases (90KD) obtained above were subjected to SDS-PAGE protein electrophoresis to identify the purity of the DNA polymerase proteins. The results are as follows: Figure 1 As shown, Figure 1 Lanes 1 / 2 / 3 / 4 contain 100 ng / 200 ng / 400 ng / 800 ng of BSA, lanes 1-5 contain KODWT DNA polymerase, lane 6 contains KODL DNA polymerase, and lane 7 contains KODW DNA polymerase. Figure 1 It can be seen that KODWT, KODL, and KODW DNA polymerases have high protein purity.

[0050] Example 3: Verification of DNA polymerase activity 1. DNA polymerase activity assay for amplifying a 6kb target band The reaction system is shown in Table 1: Table 1 reagents Final concentration 10*KOD reaction buffer (Sigma) 1*KOD reaction buffer Primer tttatgcatttctttccagacttgtt (SEQ ID NO: 7) 0.4 μM Primer cgacggagctcgaattcggatccttat (SEQ ID NO: 8) 0.4 μM Template (Pet30a plasmid) 100 ng dNTPs (10mM) 0.2 mM <![CDATA[25 mM MgSO4]]> 1.5 mM enzymes 100 ng Add water to make up the total volume 25 μL The amplification procedure is shown in Table 2: Table 2

[0051] The results are as follows Figure 2 As shown, Figure 2Images 1, 2, and 3 respectively demonstrate the amplification capabilities of KODWT, KODL, and KODW DNA polymerases for the 6Kb target band. All three polymerases can amplify the 6Kb target band under the correct primer conditions.

[0052] 2. DNA polymerase activity assay for amplifying the 28.3kb target band The reaction system is shown in Table 3: Table 3

[0053] The reaction system is shown in Table 4: Table 4

[0054] The results are as follows Figure 3 As shown, Figure 3 Tables 1, 2, and 3 respectively demonstrate the amplification capabilities of KODL, KODWT, and KODW DNA polymerases for a 30Kb target band. KODL and KODW can both amplify the 30Kb target band under the correct primer conditions, while KODWT cannot.

[0055] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A DNA polymerase mutant, characterized in that: The amino acid sequence of the DNA polymerase mutant is shown in SEQ ID NO:3 or SEQ ID NO:

5.

2. A fusion protein, characterized in that: The fusion protein includes the DNA polymerase mutant of claim 1 and a tag that assists in purification or expression.

3. The fusion protein according to claim 2, characterized in that: The tag includes at least one of His, Flag, HA, GST, GFP, and Biotin.

4. A nucleic acid molecule, characterized in that: The nucleic acid molecule expresses the DNA polymerase mutant of claim 1 or the fusion protein of any one of claims 2-3; Preferably, the nucleic acid molecule comprises a sequence as shown in SEQ ID NO: 4 or SEQ ID NO:

6.

5. A carrier, characterized in that: The carrier includes the nucleic acid molecule as described in claim 4.

6. A host cell, characterized in that: The host cell comprises the nucleic acid molecule of claim 4 or the vector of claim 5.

7. A reagent kit, characterized in that: The kit comprises the DNA polymerase mutant of claim 1 or the fusion protein of any one of claims 2-3, and reaction aids.

8. A method for preparing the DNA polymerase mutant of claim 1 or the fusion protein of any one of claims 2-3, comprising the following steps: The host cells described in claim 6 are cultured and then isolated and purified.

9. The use of the DNA polymerase mutant of claim 2 or 3, or the fusion protein of any one of claims 2 or 3, in amplifying long DNA templates.

10. A method for amplifying a long DNA template, comprising the following steps: DNA synthesis was performed using the DNA polymerase mutant of claim 1, the fusion protein of any one of claims 2-3, or the kit of claim 7.

Images