Phi29 dna polymerase mutants, methods of making and using same

Abstract

The application discloses a phi29 DNA polymerase mutant, a preparation method and application thereof, and relates to the technical field of biology.The application specifically discloses a phi29 DNA polymerase mutant which comprises at least one mutation of M8K, V54C, M97E, T203D, Y224K, R496S, E515N and S527T compared with a wild-type phi29 DNA polymerase.The phi29 DNA polymerase mutant has higher DNA binding capacity and higher thermal stability compared with the wild-type phi29 DNA polymerase, and has a good application prospect in the fields of nucleic acid amplification and sequencing.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a phi29 DNA polymerase mutant, a preparation method and application thereof. BACKGROUND

[0002] Phi29 DNA polymerase is encoded by a bacteriophage phi29 that infects Bacillus subtilis, and is widely used in nucleic acid amplification and various sequencing technologies, including multiple displacement amplification (MDA), rolling circle amplification (RCA), whole genome amplification (WGA), SNP detection, single polymerase molecule real-time DNA sequencing, DNA nanoball sequencing, and nanopore sequencing using a sequencing-by-synthesis method. However, the DNA binding ability and thermal stability of wild-type phi29 DNA polymerase are relatively poor, which often affects the yield of library construction and the sequencing effect in actual application scenarios. SUMMARY

[0003] The present application aims to at least solve one of the technical problems existing in the prior art. To this end, the present application provides a phi29 DNA polymerase mutant, which has higher DNA binding ability and thermal stability compared to wild-type phi29 DNA polymerase.

[0004] The present application also provides a biological material related to the above-mentioned phi29 DNA polymerase mutant.

[0005] The present application also provides an enzyme preparation.

[0006] The present application also provides a preparation method of the above-mentioned phi29 DNA polymerase mutant.

[0007] The present application also provides a method for amplifying DNA molecules.

[0008] The present application also provides applications related to the above-mentioned phi29 DNA polymerase mutant, biological material or enzyme preparation.

[0009] The present application also provides a kit for nucleic acid amplification or sequencing.

[0010] According to the first aspect of the present application, a phi29 DNA polymerase mutant is provided, which comprises at least one of the following mutation sites: M8K, V54C, M97E, T203D, Y224K, R496S, E515N, S527T, compared to wild-type phi29 DNA polymerase, and the amino acid sequence of the wild-type phi29 DNA polymerase is shown in SEQ ID NO: 1.

[0011] The phi29 DNA polymerase mutant according to the embodiments of the present application has at least the following beneficial effects:

[0012] Compared with the wild-type phi29 DNA polymerase, the phi29 DNA polymerase mutant of the embodiment has higher genome amplification efficiency, the brightness of the band obtained after amplification is higher than that of the wild type when single cell library is constructed by MDA technology, and the amplification efficiency is higher; and has higher thermal stability. It has good application prospect in the field of nucleic acid amplification and sequencing.

[0013] According to some embodiments of the present application, the phi29 DNA polymerase mutant has any one of A1) to A3):

[0014] A1), M8K, V54C, M97E, T203D, Y224K, R496S;

[0015] A2), M8K, M97E, Y224K, R496S, E515N, S527T;

[0016] A3), M8K, M97E, Y224K, R496S, S527T.

[0017] According to some embodiments of the present application, the phi29 DNA polymerase mutant further comprises a tag.

[0018] According to some embodiments of the present application, the tag is connected to the middle and / or N-terminal or / and C-terminal of the phi29 DNA polymerase mutant.

[0019] According to some embodiments of the present application, the tag sequence comprises at least one of the tag sequences facilitating the solubilization, purification and detection of the phi29 DNA polymerase mutant. It can be understood that the phi29 DNA polymerase mutant of the present application can comprise one or more tag sequences; multiple tag sequences can comprise a combination of multiple same tag sequences, or a combination of multiple different tag sequences. For example: the tag facilitating the solubilization of the phi29 DNA polymerase mutant includes but is not limited to nus tag sequence or maltose binding protein tag sequence; the tag facilitating the purification of the phi29 DNA polymerase mutant includes but is not limited to strep tag sequence, His tag sequence, GST tag sequence, pelB signal tag sequence or ompA signal tag sequence; the tag facilitating the detection of the phi29 DNA polymerase mutant includes but is not limited to horseradish peroxidase (HRP) tag sequence, beta-galactosidase tag sequence, luciferase tag sequence, green fluorescent protein (GFP) tag sequence, HcRed tag sequence, DsRed tag sequence or cyan fluorescent protein (CFP) tag sequence. The tag can be specifically His tag sequence.

[0020] According to the second aspect of the present application, the biological material associated with the phi29 DNA polymerase mutant as described in the first aspect of the present application is any one of B1) to B4):

[0021] B1) a nucleic acid molecule encoding the phi29 DNA polymerase mutant as described in the first aspect of the present application;

[0022] B2) an expression cassette comprising the nucleic acid molecule of B1);

[0023] B3) a recombinant vector comprising the nucleic acid molecule of B1) or the expression cassette of B2);

[0024] B4) a recombinant biological cell comprising the nucleic acid molecule of B1), the expression cassette of B2), or the recombinant vector of B3).

[0025] According to some embodiments of the present application, the nucleic acid molecule is any one of B11) to B15):

[0026] B11) a DNA molecule having the nucleotide sequence as set forth in SEQ ID NO: 6 from position 34 to 1761;

[0027] B12) a DNA molecule having the nucleotide sequence as set forth in SEQ ID NO: 7 from position 34 to 1761;

[0028] B13) a DNA molecule having the nucleotide sequence as set forth in SEQ ID NO: 8 from position 34 to 1761;

[0029] B14) a DNA molecule having 80%, 85% or 90% or more homology with the nucleotide sequence as set forth in any one of B11) to B13), and encoding the phi29 DNA polymerase mutant;

[0030] B15) a DNA molecule hybridizing to the nucleotide sequence as defined in any one of B11) to B14) under stringent conditions, and encoding the phi29 DNA polymerase mutant.

[0031] According to some embodiments of the present application, the stringent conditions can be hybridization in a solution of 2xSSC, 0.1% SDS at 68°C and washing the membrane twice for 5 min each time, or hybridization in a solution of 0.5xSSC, 0.1% SDS at 68°C and washing the membrane twice for 15 min each time.

[0032] According to some embodiments of the present application, the expression cassette refers to DNA capable of expressing the phi29 DNA polymerase mutant in a host cell. The DNA can include not only a promoter to initiate transcription of the phi29 DNA polymerase mutant gene, but also a terminator to terminate transcription of the phi29 DNA polymerase mutant gene. Further, the expression cassette can also include an enhancer sequence.

[0033] According to some embodiments of the present application, the vector can be a plasmid, a cosmid, a bacteriophage or a viral vector. For example, the PET-28a vector can be used.

[0034] According to some embodiments of the present application, the recombinant vector can be a recombinant vector obtained by inserting a DNA molecule encoding the phi29 DNA polymerase mutant into a multiple cloning site of the vector.

[0035] According to some embodiments of the present application, the biological cell includes a prokaryotic cell and a eukaryotic cell. The prokaryotic cell includes a bacterium or an alga. The eukaryotic cell includes a fungus, a mammalian cell or an insect cell. Among them, the bacterium can be E. coli, such as E. coli DH5a or E. coli BL21. The recombinant organism does not contain reproductive material.

[0036] According to some embodiments of the present application, the recombinant biological cell is a recombinant biological cell obtained by introducing B1) the nucleic acid molecule, B2) the expression cassette or B3) the recombinant vector into a biological cell. Specifically, it can be a recombinant E. coli obtained by introducing a recombinant vector into E. coli DH5a or E. coli BL21.

[0037] According to some embodiments of the third aspect of the present application, an enzyme preparation includes the phi29 DNA polymerase mutant described in the first aspect of the present application.

[0038] According to some embodiments of the present application, the enzyme preparation further includes a reaction premix.

[0039] According to some embodiments of the present application, the reaction premix includes at least one of Tris-HCl, Mg 2+ , NH4 + , DTT, dNTPs, BSA.

[0040] According to some embodiments of the present application, the NH4 + is derived from an ammonium salt. The ammonium salt includes at least one of an organic ammonium salt (including but not limited to ammonium acetate), an inorganic ammonium salt (including but not limited to (NH4)2SO4, NH4Cl or a combination thereof).

[0041] According to some embodiments of the present application, the Mg2+ The sources include at least one of magnesium chloride, magnesium acetate, and magnesium sulfate.

[0042] According to some embodiments of the present invention, the reaction premix comprises 20–100 mmol / L Tris-HCl and 9–11 mmol / L Mg. 2+ 9–11 mmol / L NH4 + 4–5 mmol / L DTT, 120–130 μmol / L dNTPs, and 100–110 ng / μL BSA. For example, the reaction premix may include 50 mmol / L Tris-HCl and 10 mmol / L Mg... 2+ 10 mmol / L NH4 + , 4mmol / L DTT, 125μmol / L dNTPs, 100ng / μL BSA.

[0043] According to some embodiments of the present invention, the pH of the reaction premix is ​​7.2 to 7.8.

[0044] It is understood that the reaction premix should preferably not affect the activity of the phi29 DNA polymerase mutant.

[0045] The method for preparing the phi29 DNA polymerase mutant according to the first aspect of the present invention, as described in the fourth aspect embodiment of the present invention, includes:

[0046] The coding gene of the phi29 DNA polymerase mutant described in the first aspect of the present invention is introduced into a biological cell to express the coding gene, thereby obtaining the phi29 DNA polymerase mutant.

[0047] According to some embodiments of the present invention, the biological cells include prokaryotic cells and eukaryotic cells.

[0048] According to some embodiments of the present invention, the prokaryotic cells include bacteria or algae. The bacteria may be *Escherichia coli* (e.g., *E. coli* BL21).

[0049] According to some embodiments of the present invention, the eukaryotic cells include fungi (such as yeast), mammalian cells (such as HEK293 cells), or insect cells.

[0050] A method for amplifying DNA molecules according to a fifth aspect embodiment of the present invention includes the following steps:

[0051] The DNA molecule was amplified using the phi29 DNA polymerase mutant described in the first aspect of the present invention.

[0052] According to some embodiments of the present application, the application can specifically include the following steps:

[0053] Mixing the phi29 DNA polymerase mutant, the DNA molecule (template DNA), primer and reaction premix, and reacting.

[0054] The use according to the sixth aspect of the present application.

[0055] According to some embodiments of the present application, the use is the use of any one of C1) to C3) in the preparation of a nucleic acid amplification or sequencing product.

[0056] C1), the phi29 DNA polymerase mutant described in the first aspect of the present application;

[0057] C2), the biological material described in the second aspect of the present application;

[0058] C3), the enzyme preparation described in the third aspect of the present application.

[0059] According to some embodiments of the present application, the use is the use of the phi29 DNA polymerase mutant described in the first aspect of the present application or the enzyme preparation described in the third aspect of the present application in nucleic acid amplification or sequencing.

[0060] According to some embodiments of the present application, the nucleic acid amplification includes but is not limited to rolling circle amplification or multiple chain displacement amplification.

[0061] According to some embodiments of the present application, the sequencing includes DNA sequencing. Including but not limited to single-cell sequencing (such as can be used as a DNA polymerase for single-cell library construction), single-polymerase molecule real-time DNA sequencing, DNA nanoball sequencing or nanopore sequencing.

[0062] A kit for nucleic acid amplification or sequencing according to the seventh aspect of the present application, comprising the above-mentioned phi29 DNA polymerase mutant or the above-mentioned enzyme preparation.

[0063] Other features and advantages of the present application will be set forth in the subsequent description, and in part will become apparent from the description, or will be learned through practice of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0064] Figure 1 A three-dimensional modeling structure diagram of the phi29 DNA polymerase mutant containing each mutation site;

[0065] Figure 2Figure 1 is a graph of the results of expression and purification of the wild-type phi29 DNA polymerase (A) and phi29 DNA polymerase mutants phi29-m1 (B), phi29-m2 (C), and phi29-m3 (D); M is a protein marker, and lanes 1-12 are samples 1-12, respectively;

[0066] Figure 3 Figure 2 is a graph of the results of relative enzyme activity detection of the wild-type phi29 DNA polymerase (A) and phi29 DNA polymerase mutants phi29-m1 (B), phi29-m2 (C), and phi29-m3 (D);

[0067] Figure 4 Figure 3 is a graph of the results of relative enzyme activity detection of the wild-type phi29 DNA polymerase (A) and phi29 DNA polymerase mutants phi29-m1 (B), phi29-m2 (C), and phi29-m3 (D) after heat treatment at 37°C for 6 hours;

[0068] Figure 5 Figure 4 is a graph of the results of genome amplification performance detection of the wild-type phi29 DNA polymerase and phi29 DNA polymerase mutants phi29-m1 to phi29-m3; M is a DNA marker (GL DNA Marker 10000), 1-2 are experimental groups with a template amount of 10 ng, 3-4 are experimental groups with a template amount of 0.1 ng, and 5 is a negative control group without enzyme addition;

[0069] Figure 6 Figure 5 is a graph of the results of genome amplification performance detection of the wild-type phi29 DNA polymerase and phi29 DNA polymerase mutant phi29-m1 after heat treatment; M is a DNA marker (GL DNA Marker 10000), 1-2 are untreated groups, 3-4 are heated at 30°C for 30 minutes, 5-6 are heated at 37°C for 30 minutes, and 7-8 are heated at 42°C for 30 minutes;

[0070] Figure 7 Figure 6 is a graph of the results of genome amplification performance detection of phi29 DNA polymerase mutant phi29-m2 and phi29 DNA polymerase mutant phi29-m3 after heat treatment; M is a DNA marker (GL DNA Marker 10000), 1-2 are untreated groups, 3-4 are heated at 30°C for 30 minutes, 5-6 are heated at 37°C for 30 minutes, and 7-8 are heated at 42°C for 30 minutes. DETAILED DESCRIPTION

[0071] The concept and the technical effects of the present application will be described clearly and completely in combination with the embodiments, so as to fully understand the purpose, features and effects of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments of the present application, other embodiments obtained by those skilled in the art without creative labor are within the protection scope of the present application.

[0072] The unspecified conditions in the embodiments are carried out according to the conventional conditions or the conditions suggested by the manufacturers. The reagents or instruments used are the conventional products which can be obtained by the market purchase, and the manufacturers are not specified.

[0073] In the description of the present application, the terms "comprising" and "having" and any variations thereof are intended to cover the non-exclusive inclusion, for example, the process, method or product including a series of steps or units does not have to be limited to the clearly listed steps or units, but can include other steps or units which are not clearly listed or inherent to these processes, methods or products.

[0074] Unless specifically defined, all the scientific or technical professional terms in the present patent are consistent with the ordinary understanding of most people in the field.

[0075] The following amino acid sequences are in the order from N-terminal to C-terminal; the nucleotide sequences are in the order from 5'-terminal to 3'-terminal.

[0076] The term "amplification" refers to the process that the number of the target nucleic acid fragments is increased under the action of nucleic acid polymerase.

[0077] In order to obtain phi29 DNA polymerase with good thermal stability and amplification performance, the wild-type phi29 DNA polymerase is mutated and screened in large quantities, and the phi29 DNA polymerase mutants phi29-m1, phi29-m2 and phi29-m3 are obtained.

[0078] The amino acid sequence of wild-type phi29 DNA polymerase phi29-mt is shown as SEQ ID NO: 1. MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDHSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIINWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDKEVRYAYRGGFTWLNDRFKEKEIGEGMVFDVNSLYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSEGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWARYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYMKEVDGKLVEGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIK (SEQ ID NO: 1).

[0079] The phi29 DNA polymerase mutant phi29-m1 is formed based on the amino acid shown as SEQ ID NO: 1, and the specific amino acid mutation is M8K, V54C, M97E, T203D, Y224K, and R496S. The amino acid sequence is specifically shown as SEQ ID NO: 2.

[0080] MKHMPRKKYSCDFETTTKVEDCRVWAYGYMNIEDHSEYKIGNSLDEFMAWVLKCQADLYFHNLKFDGAFIINWLERNGFKWSADGLPNTYNTIISREGQWYMIDICLGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFKDIIDTKKFKKVFPTLSLGLDKEVRKAYRGGFTWLNDRFKEKEIGEGMVFDVNSLYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSEGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWARYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWAHESTFKRAKYLSQKTYIQDIYMKEVDGKLVEGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIK (SEQ ID NO: 2).

[0081] The phi29 DNA polymerase mutant phi29-m2 is formed based on the amino acids shown in SEQ ID NO: 1, and the specific amino acid mutation is M8K, M97E, Y224K, R496S, E515N, S527T. The amino acid sequence is specifically shown in SEQ ID NO: 3.

[0082] MKHMPRKKYSCDFETTTKVEDCRVWAYGYMNIEDHSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIINWLERNGFKWSADGLPNTYNTIISREGQWYMIDICLGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDKEVRKAYRGGFTWLNDRFKEKEIGEGMVFDVNSLYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSEGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWARYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWAHESTFKRAKYLSQKTYIQDIYMKEVDGKLVNGSPDDYTDIKFTVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIK (SEQ ID NO: 3).

[0083] The phi29 DNA polymerase mutant phi29-m3 is formed based on the amino acids shown in SEQ ID NO: 1, and the specific amino acid mutation is M8K, M97E, Y224K, R496S, S527T. The amino acid sequence is specifically shown in SEQ ID NO: 4.

[0084] MKHMPRKKYSCDFETTTKVEDCRVWAYGYMNIEDHSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIINWLERNGFKWSADGLPNTYNTIISREGQWYMIDICLGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDKEVRKAYRGGFTWLNDRFKEKEIGEGMVFDVNSLYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSEGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWARYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWAHESTFKRAKYLSQKTYIQDIYMKEVDGKLVEGSPDDYTDIKFTVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIK (SEQ ID NO: 4).

[0085] Example 1

[0086] 1. Construction of expression vector

[0087] A DNA molecule (nucleotide sequence as shown in SEQ ID NO: 5) was synthesized by connecting 8x histidine tag at the N-terminal of wild-type phi29 DNA polymerase with a connecting peptide (amino acid sequence of GS) designed. The DNA molecule was connected with PET-28a vector to obtain expression vector PET-28a / phi29-mt.

[0088]

[0089] The primers were designed by point mutation method, and the nucleotide sequence (SEQ ID NO: 5) of wild-type phi29 DNA polymerase was subjected to point mutation, respectively. The specific primer sequences are as follows:

[0090] phi29-FP1: 5'-GCCGCGCAAAAAGTATAGCTGCGATT-3';

[0091] phi29-RP1: 5'-TCGAAATCGCAGCTATACTTTTTGCGC-3';

[0092] phi29-FP2: 5'-TGCTGAAATGCCAGGCGGATCTGTAT-3';

[0093] phi29-RP2: 5'-TCCGCCTGGCATTTCAGCACCCAC-3';

[0094] phi29-FP3: 5'-TTTCACGCGAGGGCCAGTGGTATAT-3';

[0095] phi29-RP3: 5'-ACTGGCCCTCGCGTGAAATAATGG-3';

[0096] phi29-FP4: 5'-CAAAGATATTATTGACACCAAAAAATTTAA-3';

[0097] phi29-RP4: 5'-GCTTCAAAGATATTATTGACACCAAAAAA-3';

[0098] phi29-FP5: 5'-AGTGCGCAAAGCGTATCGTGGCGG-3';

[0099] phi29-RP5: 5'-CACGATACGCTTTGCGCACTTCTTTAT-3';

[0100] phi29-FP6: 5'-CGAAATATCTGAGCCAGAAAACCTATA-3';

[0101] phi29-RP6: 5'-GTTTTCTGGCTCAGATATTTCGCACG-3';

[0102] phi29-FP7: 5'-ACTGGTGAACGGCAGCCCGGATGA-3';

[0103] phi29-RP7: 5'-GGGCTGCCGTTCACCAGTTTGCCAT-3';

[0104] phi29-FP8: 5'-TTAAATTTACCGTGAAATGCGCCGGC-3';

[0105] phi29-RP8: 5'-CATTTCACGGTAAATTTAATATCGGTG-3'.

[0106] The expression vector PET-28a / phi29-mt of the wild-type phi29 DNA polymerase was used as a template (200 ng), 25 μL of 2x ApexHF HS PCR Master Mix (Hunan Aikuo Rui Biological Engineering Co., Ltd., item number AG12209), 1 μL of upstream primer (10 μmol / L), 1 μL of downstream primer (10 μmol / L) were added, and water was added to 50 μL for PCR amplification. The PCR amplification program was: 94°C for 30 s; 98°C for 10 s, 55°C for 15 s, 72°C for 1 min, for a total of 25 cycles. The recovered PCR product was purified using a SteadyPure PCR reaction liquid purification kit (Hunan Aikuo Rui Biological Engineering Co., Ltd., item number AG21003), and the recovered product was transformed into DH5α competent cells. Single colonies were picked for colony PCR identification, and positive single clones were sequenced for verification. The correct competent cells were cultured, and plasmids were extracted to obtain recombinant expression vectors capable of expressing phi29-m1, phi29-m2, and phi29-m3, respectively.

[0107] The nucleotide sequence of the phi29 DNA polymerase mutant phi29-m1 is shown as SEQ ID NO: 6.

[0108]

[0109] The nucleotide sequence of the phi29 DNA polymerase mutant phi29-m2 is shown as SEQ ID NO: 7.

[0110]

[0111] The nucleotide sequence of the phi29 DNA polymerase mutant phi29-m3 is shown as SEQ ID NO: 8.

[0112]

[0113] 2. Expression:

[0114] The recombinant plasmid obtained in step 1 was transformed into host cell E. coli BL21, respectively, and single colonies were picked and inoculated into 100 mL LB medium containing 50 μg / mL ampicillin, and placed in a 30°C shaker for overnight culture. Then, 2 L LB medium containing 50 μg / mL ampicillin was inoculated with the culture at a volume ratio of 1:100, and cultured in a 30°C shaker until the OD 600 was 0.6-0.8. IPTG was added to a final concentration of 0.4 mmol / L, and the temperature was reduced to 16°C for continued induction for 16-18 h. The induced bacterial cells were collected by centrifugation and weighed, and the wet weight of the bacterial cells was recorded, and stored at -80°C.

[0115] 3. Purification:

[0116] The bacterial cells stored at -80°C after induction were resuspended in 5 mL lysis buffer (50 mM Tris-HCl, 1 mM EDTA, 300 mM NaCl, 1 mM DTT, pH 7.5) per gram of bacterial cells, and the bacterial cell suspension (sample 1) was obtained. The bacterial cells were lysed by a high-pressure disrupter, and the high-pressure disruption conditions were 750 bar, and the bacterial cells were disrupted for three cycles. The lysed bacterial cells were centrifuged at 12000 rpm for 30 min at 4°C, and the supernatant A (sample 3) was collected in a 200 mL sterile beaker, and the precipitate A (sample 4) was discarded. 10% PEI was added to the supernatant A to a final concentration of 0.6%, and the mixture was stirred at low speed for 30-60 min, and then centrifuged at 12000 rpm for 30 min at 4°C. The supernatant B (sample 5) was collected in a new 200 mL sterile beaker, and the precipitate B (sample 6) was discarded. (NH4)2SO4 was added to the supernatant B to a final concentration of 65%, and then centrifuged at 12000 rpm for 30 min at 4°C. The precipitate C (sample 7) was collected, and the supernatant C was discarded. The precipitate C was dissolved in buffer A (50 mM Tris-HCl, 1 mM EDTA, 300 mM NaCl, 1 mM DTT, 20 mM imidazole, pH 7.5) to obtain the purified protein solution (sample 8).

[0117] After equilibrating the column Ni-NTA Purose 6 Fast Flow (purchased from Jiaxing Qianpure Biotechnology Co., Ltd.) with buffer A, the protein solution to be purified was loaded into the column, the column was first washed with buffer A, and then eluted with buffer B (50 mM Tris-HCl, 1 mM EDTA, 300 mM NaCl, 1 mM DTT, 700 mM imidazole, pH 7.5) at a gradient of 0% to 100%. The eluate was detected by SDS-PAGE protein electrophoresis, and the eluate A containing the target protein was collected (sample 9). The eluate A was dialyzed into buffer A, and the dialysate A (sample 10) was collected. The dialysate A was subjected to secondary nickel ion affinity chromatography purification, and the steps were the same as above. The eluate B containing the target protein was collected (sample 11), and the eluate B was dialyzed into buffer A. The dialysate B (sample 12) was collected for standby use.

[0118] 30 μL of each of samples 1 to 12 was subjected to SDS-PAGE electrophoresis analysis of the purification results. The results are shown in Figure 2

[0119] Detection Example 1

[0120] Within a certain period of time, a control curve was established according to the amount of product obtained under different enzyme addition amounts of commercial DNA polymerase, and a curve of the DNA polymerase to be tested was also established. The ratio of the slopes of the two curves was calculated, which could be used to determine the enzyme activity of the DNA polymerase to be tested. A hairpin type oligonucleotide sequence was designed, and under the catalysis of DNA polymerase, dNTPs gradually penetrated, and the hairpin type oligonucleotide sequence finally formed a double-stranded DNA product. SYBR Green I is a high-sensitivity fluorescent dye that binds to double-stranded DNA and can produce a fluorescent signal when it binds to double-stranded DNA. The fluorescence signal intensity is positively correlated with the concentration of double-stranded DNA. By measuring the fluorescence signal value within a certain period of time, the enzyme activity value of the DNA polymerase to be tested can be calculated.

[0121] ​Enzyme activity assay: A hairpin oligonucleotide sequence (Test 1: 5'-tagcgaaggatgtgaacctaatcccTGCTCCCGCGGCCGatctgcCGGCCGCGGGAGCA-3') was designed. According to the brightness of the protein band obtained after SDS-PAGE electrophoresis of the purified protein, the wild-type and mutant phi29 DNA polymerases obtained by purification were pre-calculated for enzyme activity in a certain proportion to obtain the estimated enzyme activity value. The enzyme stock solution was diluted to 10 U / μL, and then the commercial DNA polymerase (purchased from Coastal Protein, product number E013-02A, as the control group), wild-type phi29 DNA polymerase solution and phi29 DNA polymerase mutant solution were diluted by different multiples using the enzyme stock solution to obtain the diluted enzyme solution. 5 μL of the diluted enzyme solution was added to 20 μL of the reaction system (25 mmol / L Tris-HCl, 50 mmol / L KCl, 5 mmol / L (NH4)2SO4, 2.5 mmol / L MgCl2, 0.1% Triton X-100, 0.25 mmol / L dNTPs, 0.5 μmol / L Test 1, pH 8.5), and the reaction was carried out at 30°C for 5 min, immediately cooled on ice, and EDTA was added to a final concentration of 10 mM to terminate the reaction; 1×SYBR dye was added at a volume ratio of 1:30, and an enzyme marker was used for detection. Note that two parallel samples were prepared for each dilution to reduce experimental error, and two blank controls were prepared for each reaction to subtract the fluorescence background. The detection of the original fluorescence signal value data was derived, the fluorescence background signal value was subtracted, and the slope value of the fluorescence signal value and the enzyme amount was calculated.

[0122] The wild-type phi29 DNA polymerase and each phi29 DNA polymerase mutant were heat-treated at 37°C for 6 h, and then the enzyme activity was determined according to the above method, and the residual enzyme activity was calculated. The corresponding wild-type phi29 DNA polymerase or phi29 DNA polymerase mutant without heat treatment was used as the untreated group.

[0123] The results are shown in FIGS. Figures 3-4 and Table 1. Among them, the enzyme activity of the wild-type phi29 DNA polymerase after heat treatment has disappeared, and the numerical value cannot form a linear relationship. As shown in FIG. Figure 4 A.

[0124] Table 1

[0125]

[0126] The phi29 DNA polymerase mutants phi29-m1, phi29-m2 and phi29-m3 have good thermal stability, and still have high enzyme activity after 6 hours of heat treatment at 37°C. The wild-type phi29 DNA polymerase loses enzyme activity and has poor thermal stability.

[0127] Detection Example 2

[0128] In this detection example, the amplification performance of single-cell library construction of the phi29 DNA polymerase mutants was detected. Human genomic DNA was used as a template, and the wild-type phi29 DNA polymerase or the phi29 DNA polymerase mutants were used for single-cell library construction amplification reaction by MDA technology. The amplification efficiency was determined by the brightness of the whole electrophoresis band obtained after the amplification reaction. The steps are as follows:

[0129] After 20 μL of the reaction system (50 mmol / L Tris-HCl, 10 mmol / L MgCl2, 10 mmol / L (NH4)2SO4, 4 mmol / L DTT, 125 μmol / L dNTPs, 25 μmol / L Random Primer 6 (purchased from NEB, item number S1230S), 100 ng / μL BSA, 10 U of enzyme, pH 7.5) containing 10 ng or 0.1 ng of template was incubated at 30°C for 2 hours, and then incubated at 65°C for 10 minutes, the product was detected by electrophoresis using a 1% agarose gel.

[0130] The results are shown in Figure 5

[0131] Under the same reaction system and reaction conditions, the band brightness obtained by the phi29 DNA polymerase mutants phi29-m1, phi29-m2 and phi29-m3 was much brighter than that of the wild-type phi29 DNA polymerase. This indicates that the amplification efficiency of the phi29 DNA polymerase mutants phi29-m1, phi29-m2 and phi29-m3 for single-cell library construction is better than that of the wild-type phi29 DNA polymerase.

[0132] Detection Example 3

[0133] In this detection example, the amplification performance of single-cell library construction of the phi29 DNA polymerase mutants after heat treatment was detected. The wild-type phi29 DNA polymerase and each phi29 DNA polymerase mutant were heat treated at 30°C, 37°C and 42°C for 30 minutes, respectively, and then the change in the amplification performance of single-cell library construction was detected. The method was the same as that in Detection Example 2 (the template addition amount was 10 ng).

[0134] The experimental results are shown in​Figures 6-7 As shown.

[0135] In the same reaction system, the whole band of wild-type phi29 DNA polymerase is weak, and only after 30 min of treatment at 30℃, there is a band, and after heat treatment at 37℃ or 42℃, there is no amplification band. The mutant phi29-m1 of phi29 DNA polymerase has very bright amplification bands after heat treatment at 30℃, 37℃ and 42℃, which is stronger than the wild-type phi29 DNA polymerase. The mutant phi29-m2 of phi29 DNA polymerase can withstand heat treatment at 30℃ and 37℃, and has very bright amplification bands after heat treatment at 30℃ and 37℃, which is stronger than the wild-type phi29 DNA polymerase. The mutant phi29-m3 of phi29 DNA polymerase can withstand heat treatment at 30℃, and the whole band brightness is still stronger than the wild-type phi29 DNA polymerase. This shows that the thermal stability of the mutants phi29-m1, phi29-m2 and phi29-m3 of phi29 DNA polymerase is improved compared with the wild-type phi29 DNA polymerase.

[0136] The embodiments of the present application are described in detail above in combination with the drawings, but the present application is not limited to the above embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the purpose of the present application.

Claims

1. A mutant of phi29 DNA polymerase, characterized in that, The amino acid sequence of the wild-type phi29 DNA polymerase is shown in SEQ ID NO: 1; the phi29 DNA polymerase mutant has a mutation site selected from any one of A1) to A3) as compared with the wild-type phi29 DNA polymerase: A1), M8K, V54C, M97E, T203D, Y224K, R496S; A2), M8K, M97E, Y224K, R496S, E515N, S527T; A3), M8K, M97E, Y224K, R496S, S527T.

2. The phi29 DNA polymerase mutant according to claim 1, characterized in that, The phi29 DNA polymerase mutant further comprises a tag.

3. A biological material associated with the phi29 DNA polymerase mutant of claim 1 or 2, characterized by: The biological material is any one of B1) to B4): B1), a nucleic acid molecule encoding the phi29 DNA polymerase mutant of claim 1 or 2; B2), an expression cassette comprising the nucleic acid molecule of B1); B3), a recombinant vector comprising the nucleic acid molecule of B1) or the expression cassette of B2); B4), a recombinant biological cell comprising the nucleic acid molecule of B1), the expression cassette of B2), or the recombinant vector of B3).

4. The biomaterial of claim 3, wherein, The nucleic acid molecule is any one of B11) to B15): B11), a DNA molecule having a nucleotide sequence as shown in SEQ ID NO: 6, positions 34 to 1761; B12), a DNA molecule having a nucleotide sequence as shown in SEQ ID NO: 7, positions 34 to 1761; B13), a DNA molecule having a nucleotide sequence as shown in SEQ ID NO: 8, positions 34 to 1761; B14), a DNA molecule having a nucleotide sequence having 80%, 85% or 90% homology or more to the nucleotide sequence shown in any one of B11) to B13), and encoding the phi29 DNA polymerase mutant; B15), a DNA molecule hybridizing to the nucleotide sequence defined in any one of B11) to B14) under stringent conditions, and encoding the phi29 DNA polymerase mutant.

5. An enzyme preparation, characterized in that, The phi29 DNA polymerase mutant of claim 1 or 2.

6. A method for preparing the phi29 DNA polymerase mutant of claim 1 or 2, characterized by, The use of the phi29 DNA polymerase mutant of claim 1 or 2 or the enzyme preparation of claim 5 for amplifying the DNA molecule. The use of the phi29 DNA polymerase mutant of claim 1 or 2 or the enzyme preparation of claim 5 for amplifying the DNA molecule.

7. A method of amplifying a DNA molecule, characterized by, 8. The use of any one of C1) to C3) in the preparation of a nucleic acid amplification or sequencing product; C1), the phi29 DNA polymerase mutant of claim 1 or 2; C2), the biological material of claim 3 or 4; C3), the enzyme preparation of claim 5.

9. The use of the phi29 DNA polymerase mutant of claim 1 or 2 or the enzyme preparation of claim 5 in nucleic acid amplification or sequencing. The use of the phi29 DNA polymerase mutant of claim 1 or 2 or the enzyme preparation of claim 5. ​ 10. A kit for nucleic acid amplification or sequencing, characterized in that, ​

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