DNA polymerase mutant for improving amplification efficiency of high GC sample and application of DNA polymerase mutant
By performing site-directed mutagenesis on DNA polymerase, KODG and KODC mutants were prepared, solving the problem of amplification failure in high GC regions and achieving efficient amplification of DNA templates with high GC content, thus expanding the application range of DNA polymerase.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-01-26
- Publication Date
- 2026-04-10
AI Technical Summary
Existing DNA polymerases are difficult to completely denature when amplifying DNA templates with high GC content due to their high thermostability, leading to amplification failure and making it impossible to effectively amplify high GC regions.
By performing site-directed mutagenesis on DNA polymerase, KODG and KODC mutants were prepared to enhance their amplification ability in high GC regions. Combined with appropriate purification and expression strategies, highly efficient DNA polymerase mutants were prepared.
It significantly improved the amplification efficiency of DNA templates with high GC content, successfully amplified a 3kb DNA fragment with 95% GC content that was difficult to amplify, and expanded the application range of DNA polymerase in complex template amplification.
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Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of biotechnology, and particularly relates to a DNA polymerase mutant for improving the amplification efficiency of high GC sample and application thereof. BACKGROUND
[0002] DNA polymerase is responsible for the replication and maintenance of the genome, which is essential for accurately transmitting genetic information from generation to generation. For high GC DNA, because GC base pairs are connected by three hydrogen bonds, while AT base pairs have only two, the double-stranded DNA (dsDNA) of GC-rich regions has very high thermal stability and high melting temperature (Tm). In the denaturation step of PCR (usually 94-98℃), these high GC regions may not be completely denatured and still maintain local double-stranded or hairpin structures, which may prevent primers from binding to the template and polymerase from synthesizing. Therefore, how to improve the extension activity of DNA polymerase to enable it to be used for the amplification of high GC content double-stranded DNA is of great importance for disease detection and biological identification. SUMMARY
[0003] The first aspect of the present application aims to provide a DNA polymerase mutant.
[0004] The second aspect of the present application aims to provide a fusion protein.
[0005] The third aspect of the present application aims to provide a nucleic acid molecule.
[0006] The fourth aspect of the present application aims to provide a vector.
[0007] The fifth aspect of the present application aims to provide a host cell.
[0008] The sixth aspect of the present application aims to provide a kit.
[0009] The seventh aspect of the present application aims to provide a preparation method.
[0010] The eighth aspect of the present application aims to provide an application.
[0011] The ninth aspect of the present application aims to provide a method for amplifying high GC content DNA template.
[0012] In order to achieve the above-mentioned purposes of the present application, the technical solutions adopted by the present application are as follows: The first aspect of the present application provides a DNA polymerase mutant, wherein the amino acid sequence of the DNA polymerase mutant is shown in SEQ ID NO: 3 or SEQ ID NO: 5.
[0013] In some embodiments of the present application, the DNA polymerase mutant comprises KODG and KODC.
[0014] wherein KODG is mutated from P to H at the 94th amino acid of KODWT; and KODC is mutated from Q to M at the 242nd amino acid of KODWT.
[0015] In a second aspect of the present application, a fusion protein is provided, which comprises the DNA polymerase mutant of the first aspect of the present application and a tag for facilitating purification or expression.
[0016] In some embodiments of the present application, the tag comprises at least one of His, Flag, HA, GST, GFP, and Biotin.
[0017] In some embodiments of the present application, the tag is located at the N-terminus and / or C-terminus of the DNA polymerase mutant.
[0018] In a third aspect of the present application, a nucleic acid molecule is provided, which expresses the DNA polymerase mutant of the first aspect of the present application or the fusion protein of the second aspect of the present application.
[0019] In some embodiments of the present application, the nucleic acid molecule comprises a sequence as set forth in SEQ ID NO: 4 or SEQ ID NO: 6.
[0020] In a fourth aspect of the present application, a vector is provided, which comprises the nucleic acid molecule of the third aspect of the present application.
[0021] In some embodiments of the present application, the vector comprises a eukaryotic cell vector or a prokaryotic cell vector.
[0022] In a fifth aspect of the present application, a host cell is provided, which comprises the nucleic acid molecule of the third aspect of the present application or the vector of the fourth aspect of the present application.
[0023] In some embodiments of the present application, the host cell comprises a eukaryotic cell or a prokaryotic cell.
[0024] In some embodiments of the present application, the eukaryotic cell comprises a yeast cell.
[0025] In some embodiments of the present application, the prokaryotic cell comprises Escherichia coli.
[0026] In a sixth aspect of the present application, a kit is provided, which comprises the DNA polymerase mutant of the first aspect of the present application or the fusion protein of the second aspect of the present application, and a reaction auxiliary reagent.
[0027] In some embodiments of the present application, the reaction auxiliary reagent includes conventional materials for DNA amplification such as reaction buffer, metal ions, dNTP, etc.
[0028] In a seventh aspect of the present application, a method for preparing the DNA polymerase mutant of the first aspect of the present application or the fusion protein of the second aspect of the present application is provided, comprising the following steps: Culturing the host cell of the fifth aspect of the present application, and isolating and purifying the DNA polymerase mutant or the fusion protein.
[0029] Specifically, the method for preparing the DNA polymerase comprises the following steps: 1) providing a gene expression sequence for expressing the DNA polymerase; 2) connecting the gene expression sequence to a gene expression vector pET-21a; 3) transforming the gene expression vector into a host cell BL21 to obtain a recombinant cell; 4) inducing expression of the recombinant cell, and collecting the bacterial body; 5) ultrasonically breaking the resuspended bacterial body, and collecting the supernatant; 6) purifying the supernatant by using a Ni 2+ affinity column, and collecting the purified protein; 7) concentrating the protein of step 6 by using an ultrafiltration column to obtain the DNA polymerase.
[0030] In some embodiments of the present application, the temperature for culturing the host cell is 20℃ 40℃; preferably 25℃ 37℃.
[0031] In an eighth aspect of the present application, the DNA polymerase mutant of the first aspect of the present application or the fusion protein of the second aspect of the present application is used for amplifying a high GC content DNA template.
[0032] In some embodiments of the present application, the high GC content refers to a GC content > 60%.
[0033] In a ninth aspect of the present application, a method for amplifying a high GC content DNA template is provided, comprising the following steps: Using the DNA polymerase mutant of the first aspect of the present application or the fusion protein of the second aspect of the present application or the kit of the sixth aspect of the present application for DNA synthesis.
[0034] The present application has the following beneficial effects: This invention utilizes site-directed mutagenesis to obtain DNA polymerase mutants KODG and KODC. While retaining the ability to amplify DNA templates with conventional GC content, it significantly improves the amplification efficiency for DNA templates with high GC content (>60%). It successfully achieves effective amplification of fragments that are difficult to amplify, such as 3kb and 95% GC content. This overcomes the technical bottleneck of existing polymerases that fail to amplify in high GC regions due to high thermostability and difficulty in complete denaturation. Thus, it expands the application range of DNA polymerase in complex template amplification and provides a more reliable enzymatic tool for the detection and identification of high GC content samples. 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 2kb target band with 50% GC content.
[0037] Figure 3 Results of an activity experiment for DNA polymerase to amplify a 3kb target band with 95% GC content. 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 KODG and KODC.
[0040] KODG is modified from P to H at the 94th amino acid position of KODWT.
[0041] KODC is mutated from Q to M at amino acid position 242 of KODWT.
[0042] The amino acid sequence of the DNA polymerase KODWT is as follows: MILDTDYITEDGKPVIRIFKKENGEFKIEYDRTFEPYFYALLKDDSAIEEVKKITAERHGTVVTVKRVEKVQKKFLGRPVEVWKLYFTHPQDVPAIRDKIREHPAVIDIYEYDIPFAKRYLIDKGLVPMEGDEELKMLAFDIETLYHEGEEFAEGPILMISYADEEGARVITWKNVDLPYVDVVSTEREMIKRFLRVVKEKDPDVLITYNGDNFDFAYLKKRCEKLGINFALGRDGSEPKIQRMGDRFAVEVKGRIHFDLYPVIRRTINLPTYTLEAVYEAVFGQPKEKVYAEEITTAWETGENLERVARYSMEDAKVTYELGKEFLPMEAQLSRLIGQSLWDVSRSSTGNLVEWFLLRKAYERNELAPNKPDEKELARRRQSYEGGYVKEPERGLWENIVYLDFRSLYPSIIITHNVSPDTLNREGCKEYDVAPQVGHRFCKDFPGFIPSLLGDLLEERQKIKKKMKATIDPIERKLLDYRQRAIKILANSYYGYYGYARARWYCKECAESVTAWGREYITMTIKEIEEKYGFKVIYSDTDGFFATIPGADAETVKKKAMEFLKYINAKLPGALELEYEGFYKRGFFVTKKKYAVIDEEGKITTRGLEIVRRDWSEIAKETQARVLEALLKDGDVEKAVRIVKEVTEKLSKYEVPPEKLVIHEQITRDLKDYKATGPHVAVAKRLAARGVKIRPGTVISYIVLKGSGRIGDRAIPFDEFDPTKHKYDAEYYIENQVLPAVERILRAFGYRKEDLRYQKTRQVGLSAWLKPKGTLE (SEQ ID NO: 1).
[0043] The nucleotide sequence of DNA polymerase KODWT is:
[0044] The amino acid sequence of DNA polymerase KODG is as follows: MILDTDYITEDGKPVIRIFKKENGEFKIEYDRTFEPYFYALLKDDSAIEEVKKITAERHGTVVTVKRVEKVQKKFLGRPVEVWKLYFTHPQDV H AIRDKIREHPAVIDIYEYDIPFAKRYLIDKGLVPMEGDEELKMLAFDIETLYHEGEEFAEGPILMISYADEEGARVITWKNVDLPYVDVVSTEREMIKRFLRVVKEKDPDVLITYNGDNFDFAYLKKRCEKLGINFALGRDGSEPKIQRMGDRFAVEVKGRIHFDLYPVIRRTINLPTYTLEAVYEAVFGQPKEKVYAEEITTAWETGENLERVARYSMEDAKVTYELGKEFLPMEAQLSRLIGQSLWDVSRSSTGNLVEWFLLRKAYERNELAPNKPDEKELARRRQSYEGGYVKEPERGLWENIVYLDFRSLYPSIIITHNVSPDTLNREGCKEYDVAPQVGHRFCKDFPGFIPSLLGDLLEERQKIKKKMKATIDPIERKLLDYRQRAIKILANSYYGYYGYARARWYCKECAESVTAWGREYITMTIKEIEEKYGFKVIYSDTDGFFATIPGADAETVKKKAMEFLKYINAKLPGALELEYEGFYKRGFFVTKKKYAVIDEEGKITTRGLEIVRRDWSEIAKETQARVLEALLKDGDVEKAVRIVKEVTEKLSKYEVPPEKLVIHEQITRDLKDYKATGPHVAVAKRLAARGVKIRPGTVISYIVLKGSGRIGDRAIPFDEFDPTKHKYDAEYYIENQVLPAVERILRAFGYRKEDLRYQKTRQVGLSAWLKPKGTLE (SEQ ID NO: 3).
[0045] The nucleotide sequence of DNA polymerase KODG is as follows:
[0046] The amino acid sequence of DNA polymerase KODC is as follows: MILDTDYITEDGKPVIRIFKKENGEFKIEYDRTFEPYFYALLKDDSAIEEVKKITAERHGTVVTVKRVEKVQKKFLGRPVEVWKLYFTHPQDVPAIRDKIREHPAVIDIYEYDIPFAKRYLIDKGLVPMEGDEELKMLAFDIETLYHEGEEFAEGPILMISYADEEGARVITWKNVDLPYVDVVSTEREMIKRFLRVVKEKDPDVLITYNGDNFDFAYLKKRCEKLGINFALGRDGSEPKI M RMGDRFAVEVKGRIHFDLYPVIRRTINLPTYTLEAVYEAVFGQPKEKVYAEEITTAWETGENLERVARYSMEDAKVTYELGKEFLPMEAQLSRLIGQSLWDVSRSSTGNLVEWFLLRKAYERNELAPNKPDEKELARRRQSYEGGYVKEPERGLWENIVYLDFRSLYPSIIITHNVSPDTLNREGCKEYDVAPQVGHRFCKDFPGFIPSLLGDLLEERQKIKKKMKATIDPIERKLLDYRQRAIKILANSYYGYYGYARARWYCKECAESVTAWGREYITMTIKEIEEKYGFKVIYSDTDGFFATIPGADAETVKKKAMEFLKYINAKLPGALELEYEGFYKRGFFVTKKKYAVIDEEGKITTRGLEIVRRDWSEIAKETQARVLEALLKDGDVEKAVRIVKEVTEKLSKYEVPPEKLVIHEQITRDLKDYKATGPHVAVAKRLAARGVKIRPGTVISYIVLKGSGRIGDRAIPFDEFDPTKHKYDAEYYIENQVLPAVERILRAFGYRKEDLRYQKTRQVGLSAWLKPKGTLE (SEQ ID NO: 5).
[0047] The nucleotide sequence of DNA polymerase KODC 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 reaches 1, the bacterial culture is removed and cooled at 4°C. After cooling, IPTG is added to a final concentration of 0.6 mM for induction, and the culture is continued at 20°C for 24 hours. The fusion polymerase is purified by Ni column affinity chromatography, and the polymerase is concentrated using an AMICON ULTRA 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, KODG, and KODC DNA polymerase (90KD) obtained above were analyzed by SDS-PAGE protein electrophoresis to determine the purity of the DNA polymerase protein. The results are as follows: Figure 1 As shown in the diagram, lanes 1-1 / 2 / 3 / 4 contain 100 ng / 200 ng / 400 ng / 800 ng of BSA, lanes 1-5 contain KODWT DNA polymerase, lanes 1-6 contain KODG DNA polymerase, and lanes 1-7 contain KODC DNA polymerase. From... Figure 1 It can be seen that the KODWT, KODG, and KODC DNA polymerases have high protein purity.
[0050] Example 3: Verification of DNA polymerase activity 1. DNA polymerase activity assay for amplifying a 2kb target band with 50% GC content. The reaction system is shown in Table 1: Table 1 Reagent Final concentration 10* KOD reaction buffer (Sigma) 1* KOD reaction buffer Primer ttagaaaaactcatcgagcatca (SEQ ID NO: 7) 0.4 μΜ Primer ggtgaaaacctctgacacat (SEQ ID NO: 8) 0.4 μΜ Template (Pet30a plasmid) 100 ng dNTPs (10 mM) 0.2 mM MgSO4 1.5 mM Enzyme 100 ng Total volume Water up to 25 μΐ The amplification procedure is shown in Table 2: Table 2
[0051] The results are as follows Figure 2 As shown, Figure 2Tables 1, 2, and 3 respectively demonstrate the amplification capabilities of KODWT, KODG, and KODC DNA polymerases for a 2Kb target band with 50% GC content. All three polymerases can amplify the 2Kb target band under the correct primer conditions.
[0052] 2. DNA polymerase activity assay for amplifying a 3kb target band with 95% GC content. The reaction system is shown in Table 3: Table 3 Reagent Units 10* KOD reaction buffer 5 μΐ 2 mM dNTPs 10 μΐ Polymerases 100 ng Template (NA09237) 15 ng Primer F (90 μΜ) TCAGGCGCTCAGCTCCGTTTCGGTTTCA (SEQ ID NO: 9) 2 μΜ Primer R (90 μΜ) AAGCGCCATTGGAGCCCCGCACTTCC (SEQ ID NO: 10) 2 μΜ MgSO4 1.5 mM ddH2O Up to 25 μΐ The reaction system is shown in Table 4: Table 4
[0053] The results are as follows Figure 3 As shown, Figure 3 Tables 1, 2, and 3 respectively demonstrate the amplification capabilities of KODWT, KODG, and KODC DNA polymerases for a 3Kb target band with 95% GC content. KODG and KODC can both amplify the 3Kb target band under the correct primer conditions, while KODWT cannot.
[0054] 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 DNA templates with high GC content.
10. A method for amplifying a DNA template with high GC content, 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.