DNA synthesis error correction enzyme and its application

Abstract

The invention discloses a DNA synthesis error correction enzyme and its application, belonging to the field of gene engineering technology. The enzyme is formed by connecting and fusing a domain of Taq DNA polymerase that catalyzes DNA synthesis, an error correction active domain of an enzyme that corrects DNA mismatches, and a functional domain of MutS protein that specifically recognizes DNA double-strand mismatches, respectively, using a flexible linker. The DNA synthesis error correction enzyme has three functions of DNA catalysis synthesis, specific recognition and repair of mismatched DNA double strands, and reduces the base error rate to less than 10 ‑3 , has good error correction efficiency and cloning accuracy, and eliminates the separate error correction step in DNA artificial synthesis, thereby increasing the efficiency of DNA artificial synthesis.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a DNA synthesis error-correcting enzyme and its application. Background Art

[0002] Artificial DNA synthesis is a key underlying technology that promotes the development of life science and related fields. Conventional genetic manipulation techniques can only make limited changes to existing sequences, while artificial DNA synthesis can improve our ability to understand, predict and manipulate life.

[0003] Synthesis errors in DNA artificial synthesis can seriously affect the assembly of gene fragments and subsequent modification. Data show that the DNA replication error rate in prokaryotic and eukaryotic cells is 10 -7 -10 -8 Artificially synthesized DNA does not have the guarantee of various mismatch repair mechanisms in the body, so the fidelity is not high, and the error rate is as high as 10 -2 -10 -3 Nucleotide insertion, deletion, and substitution errors in double-stranded DNA fragments after complementary pairing are mainly manifested as mismatches and bulges. The removal of these errors is more likely to rely on DNA enzymatic error correction technology developed based on the DNA repair system in organisms, and then use enzymes with mismatch binding or mismatch cleavage activity to correct DNA double strands, thereby enriching the correct sequence.

[0004] In order to reduce the error rate of DNA artificial synthesis and improve the efficiency, a DNA synthesis error correction enzyme and its application are proposed. Summary of the invention

[0005] The purpose of the present invention is to provide a DNA synthesis error correction enzyme and its application to solve the problems in the background technology.

[0006] The purpose of the present invention can be achieved through the following technical solutions:

[0007] A DNA synthesis error correction enzyme is formed by connecting and fusion of a domain catalyzing DNA synthesis in Taq DNA polymerase (Taq DNA polymerase), an error correction active domain in an enzyme for correcting DNA mismatches, and a functional domain specifically recognizing DNA double-strand mismatches in MutS protein (Thermus aquaticusMuts), respectively, using flexible linkers. The DNA synthesis error correction enzyme includes Taq DNA polymerase-linker-T7 EndonucleaseⅠ-linker-Thermusaquaticus Muts and Taq DNA polymerase-linker-ExonucleaseⅢ-linker-Thermusaquaticus Muts.

[0008] Furthermore, the amino acid sequence of the flexible linker is GGGGSGGGGSGGGGS.

[0009] Furthermore, enzymes for correcting DNA mismatches include T7 endonuclease I, endonuclease, V8-oxoguanine DNA glycosylase, endonuclease III, uracil-DNA glycosylase, endonuclease VIII, topoisomerase I, T4 pyrimidine dimer glycosylase, human alkyladenine DNA glycosylase and endonuclease IV.

[0010] Furthermore, the amino acid sequence of Taq DNA polymerase-linker-T7 EndonucleaseⅠ-linker-Thermusaquaticus Muts is shown in SEQ ID NO: 1, and is as follows:

[0011]

[0012] Furthermore, the amino acid sequence of Taq DNA polymerase-linker-Exonuclease III-linker-Thermusaquaticus Muts is shown in SEQ ID NO: 2, which is as follows:

[0013]

[0014] The above DNA synthesis error correction enzyme is used in artificial DNA synthesis.

[0015] Beneficial effects of the present invention:

[0016] The DNA synthesis error correction enzyme in the present invention is a new fusion protein formed by connecting the domain of Taq DNA polymerase that catalyzes DNA synthesis, the error correction active domain in the enzyme that corrects DNA mismatches, and the functional domain of MutS protein that specifically recognizes mismatched DNA double strands using a flexible linker. The DNA synthesis error correction enzyme has three functions of DNA catalysis, specific recognition and repair of mismatched DNA double strands, making the base error rate less than 10 -3 , has good error correction efficiency and cloning accuracy, and eliminates the separate error correction step in DNA artificial synthesis, thereby improving the efficiency of DNA artificial synthesis. DETAILED DESCRIPTION

[0017] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0018] Example 1

[0019] Preparation of Taq DNA polymerase-linker-T7 EndonucleaseⅠ-linker-Thermusaquaticus Muts, comprising the following steps:

[0020] Step 1: Search the PUBMED database for the DNA sequence of the domain that catalyzes DNA synthesis in Taq DNA polymerase, the DNA sequence of the DNA mismatch correction active domain in T7 nuclease I, and the DNA sequence of the functional domain that specifically recognizes DNA double-strand mismatches in MutS protein, linearly connect the three with a flexible linker, and introduce restriction sites NdeI and XhoI at their N-terminus and C-terminus, respectively, to synthesize the recombinant sequence 1, and then insert the recombinant sequence 1 into the expression vector pET-22b(+) to obtain the recombinant plasmid pET-22b(+)-1, and then transform the recombinant plasmid pET-22b(+)-1 into the Escherichia coli cloning strain DH5α.

[0021] Step 2: Pick the DH5α monoclonal strain containing the recombinant plasmid pET-22b(+)-1, inoculate it into LB medium, place it in a shaker, and culture it at 220 r / min and 37°C for 3 h to make the bacteria in the logarithmic growth phase, lower the temperature of the shaker to 28°C, and induce the expression of the target protein with IPTG (isopropylthio-β-D-galactoside) at a final concentration of 0.4 mmol / L. Centrifuge at 4000 r / min for 20 min, collect the bacteria, and then add lysozyme at a concentration of 300 mg / mL, lyse on ice for 30 min, ultrasonically break, centrifuge, collect the supernatant, purify the supernatant by magnetic beads, elute and collect the purified protein to obtain Taq DNA polymerase-linker-T7 EndonucleaseⅠ-linker-Thermusaquaticus Muts.

[0022] The DNA sequence of recombinant sequence 1 is as follows:

[0023]

[0024] The amino acid sequence of Taq DNA polymerase-linker-T7 EndonucleaseⅠ-linker-Thermus aquaticusMuts is shown in SEQ ID NO:2.

[0025] Example 2

[0026] Preparation of Taq DNA polymerase-linker-Exonuclease III-linker-Thermus aquaticus Muts, comprising the following steps:

[0027] Step 1: Search the PUBMED database for the DNA sequence of the domain that catalyzes DNA synthesis in Taq DNA polymerase, the DNA sequence of the DNA mismatch correction active domain in nuclease III, and the DNA sequence of the functional domain that specifically recognizes DNA double-strand mismatches in the MutS protein, linearly connect the three with a flexible linker, and introduce restriction sites NdeI and XhoI at their N-terminus and C-terminus, respectively, to synthesize the recombinant sequence 2, and then insert the recombinant sequence 2 into the expression vector pET-22b(+) to obtain the recombinant plasmid pET-22b(+)-2, and then transform the recombinant plasmid pET-22b(+)-2 into the Escherichia coli cloning strain DH5α.

[0028] Step 2: Pick the DH5α monoclonal strain containing the recombinant plasmid pET-22b(+)-2, inoculate it into LB medium, place it in a shaker, and culture it at 220 r / min and 37°C for 3 hours to make the bacteria in the logarithmic growth phase, lower the temperature of the shaker to 28°C, and induce the expression of the target protein with IPTG (isopropylthio-β-D-galactoside) at a final concentration of 0.4 mmol / L. Centrifuge at 4000 r / min for 20 minutes, collect the bacteria, and then add lysozyme at a concentration of 300 mg / mL, lyse on ice for 30 minutes, ultrasonically break, centrifuge, collect the supernatant, purify the supernatant by magnetic beads, elute and collect the purified protein to obtain Taq DNA polymerase-linker-ExonucleaseⅢ-linker-Thermus aquaticus Muts.

[0029] The DNA sequence of recombinant sequence 2 is as follows:

[0030]

[0031] The amino acid sequence of Taq DNA polymerase-linker-Exonuclease III-linker-Thermus aquaticus Muts is shown in SEQ ID NO: 1.

[0032] Example 3

[0033] 1. Design and synthesize 4 oligonucleotides for preparing mismatched and completely correct templates. Select oligonucleotides A1B1 and A2B2, and re-denature and anneal to form A:A mismatched double strands and T:G mismatched double strands, respectively.

[0034] The 6 oligonucleotides are shown in Table 1:

[0035] Table 1

[0036]

[0037] The oligonucleotide annealing buffer denaturation system of A1B1 and A2B2 is shown in Table 2:

[0038] Table 2

[0039] Reagents Dosage 5× DNA Oligonucleotide Annealing Buffer 10μL Nuclease-free water 30μL Oligonucleotide upper strand (100 μM) 5μL Oligonucleotide bottom strand (100 μM) 5μL

[0040] Reaction conditions: 95°C, 2 min; slowly decrease from 95°C to 25°C, with a cooling rate of 1°C / 0.8s; store at 4°C.

[0041] 2. Take 250 ng of mismatched double strands respectively, make up to 10 μL with Binding buffer, and denature and anneal again. The reaction procedure is: 98℃, 2 min; 4℃, 5 min; 37℃, 5 min; 4℃, store.

[0042] The DNA synthesis error correction enzymes prepared in Example 1 and Example 2 were respectively incubated with the double strands containing the mismatch site for 5 minutes, enriched, and the DNA supernatant was aspirated for a round of PCR amplification. The reaction system is shown in Table 3:

[0043] Table 3

[0044]

[0045] 3. Separate the target fragment from the PCR product by agarose gel electrophoresis, then recover the target fragment with a gel recovery kit and sequence it with a 3730XL gene sequencer. After sequencing, refer to the template sequence and use Seqman software to compare the sequencing results, and no base mismatch was found.

[0046] Example 4

[0047] A 1278 bp viral sequence was selected and used as a template for synthesis, and the error correction enzyme prepared in Example 1 and Example 2 was used for synthesis and error correction as the test group; Taq DNA polymerase was used for synthesis and no error correction enzyme was used for error correction, and Taq DNA polymerase was used for synthesis and T7 nuclease I was used for error correction as the control group; then amplification, recovery, transformation and cloning were performed, and sequencing was performed using Seqman software, and the error type, number of incorrect bases, etc. were counted. The sequencing results are shown in Table 4:

[0048] Table 4

[0049]

[0050] It can be seen from Table 4 that the error correction enzymes prepared in Example 1 and Example 2 have good error correction efficiency, with a base error rate of <10 -3 , the cloning accuracy rate can reach 80%.

[0051] The application of the error-correcting enzyme does not require a separate error-correcting step, thereby increasing the efficiency of artificial DNA synthesis.

[0052] It should be noted that, in this article, terms such as "comprises", "includes" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or apparatus.

[0053] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A DNA synthesis error-correcting enzyme, characterized in that The amino acid sequence of the DNA synthesis error correction enzyme is shown in SEQ ID NO: 1 or SEQ ID NO:

2.

2. Use of the DNA synthesis error correcting enzyme according to claim 1 in artificial DNA synthesis.

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