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CN116178563B - DNA synthesis error correction enzyme and its application - Google Patents

DNA synthesis error correction enzyme and its application Download PDF

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CN116178563B
CN116178563B CN202210877660.2A CN202210877660A CN116178563B CN 116178563 B CN116178563 B CN 116178563B CN 202210877660 A CN202210877660 A CN 202210877660A CN 116178563 B CN116178563 B CN 116178563B
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dna
error correction
synthesis
enzyme
dna synthesis
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CN116178563A (en
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李森
张伦
雍德祥
吴志荷
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Anhui Global Gene Technology Co ltd
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Abstract

本发明公开了一种DNA合成纠错酶及应用，属于基因工程技术领域，是将Taq DNA聚合酶中催化DNA合成的结构域、纠正DNA错配用酶中的纠错活性结构域以及MutS蛋白中特异识别DNA双链错配的功能结构域，分别利用柔性linker连接融合而成；该DNA合成纠错酶同时具有DNA催化合成、特异性识别及修复错配DNA双链三种功能，使碱基错误率＜10‑3，具有较好的纠错效率和克隆正确率，并且省去了DNA人工合成中单独的纠错步骤，增加了DNA人工合成的效率。The invention discloses a DNA synthesis error correction enzyme and application, belonging to the technical field of genetic engineering. The enzyme is formed by connecting and fusion of a domain for catalyzing DNA synthesis in Taq DNA polymerase, an error correction active domain in an enzyme for correcting DNA mismatch, and a functional domain for specifically recognizing DNA double-strand mismatch in MutS protein by using flexible linkers. The DNA synthesis error correction enzyme has three functions of DNA catalytic synthesis, specific recognition and repair of mismatched DNA double strands, so that the base error rate is 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

DNA synthesis error correction enzyme and application thereof
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to DNA synthesis error correction enzyme and application thereof.
Background
DNA synthesis is a key underlying technology that drives the development of life sciences and related fields. Conventional genetic manipulation techniques can only provide limited adaptation to existing sequences, while DNA synthesis can improve our ability to understand, predict and manipulate living organisms.
The synthetic errors generated in the artificial synthesis of DNA can seriously affect the assembly and subsequent transformation of gene fragments, and data show that the DNA replication error rate in prokaryotic and eukaryotic cells is between 10 -7-10-8, and the artificial synthesis of DNA has no guarantee of various mismatch repair mechanisms in vivo, so the fidelity is not high, and the error rate is as high as 10 -2-10-3. Nucleotide insertion, deletion and substitution errors of the double-stranded DNA fragment after complementary pairing mainly show mismatch, bulge and the like, more removal of the errors is realized by utilizing a DNA enzymatic error correction technology developed based on a DNA repair system in organisms, and then the DNA double-stranded is subjected to error correction by utilizing enzymes with mismatch binding or mismatch cutting activity, so that correct sequences are enriched.
In order to reduce the error rate of DNA artificial synthesis and improve the efficiency, a DNA synthesis error correction enzyme and application thereof are provided.
Disclosure of Invention
The invention aims to provide a DNA synthesis error correction enzyme and application thereof, which are used for solving the problems in the background technology.
The aim of the invention can be achieved by the following technical scheme:
A DNA synthesis error correction enzyme is formed by respectively utilizing flexible linker connection and fusion of a domain catalyzing DNA synthesis in Taq DNA polymerase (Taq DNA polymerase), an error correction active domain in enzyme for correcting DNA mismatch and a functional domain for specifically recognizing DNA double-chain mismatch in MutS protein (Thermus aquaticus Muts), wherein the DNA synthesis error correction enzyme comprises Taq DNA polymerase-linker-T7 EndonucleaseI-linker-Thermus aquaticus Muts and Taq DNA polymerase-linker-ExonucleaseIII-linker-Thermus aquaticus Muts.
Further, the amino acid sequence of the flexible linker is GGGGSGGGGSGGGGS.
Further, the enzymes for correcting DNA mismatches include T7 endonuclease I (T7 EndonucleaseI), endonuclease, V8-oxoguanine DNA glycosylase, endonuclease III (ExonucleaseIII), uracil-DNA glycosylase, endonuclease VIII, topoisomerase I, T4 pyrimidine dimer glycosylase, human alkyl adenine DNA glycosylase and endonuclease IV.
Further, the amino acid sequence of Taq DNA polymerase-linker-T7 EndonucleaseI-linker-Thermus aquaticus Muts is shown in SEQ ID NO.1, and is specifically as follows:
MRGMLPLFEPKGRVLLVDGHHLAYRTFHALKGLTTSRGEPVQAVYGFAKSLLKALKEDGDAVIVVFDAKAPSFRHEAYGGYKAGRAPTPEDFPRQLALIKELVDLLGLARLEVPGYEADDVLASLAKKAEKEGYEVRILTADKDLYQLLSDRIHVLHPEGYLITPAWLWEKYGLRPDQWADYRALTGDESDNLPGVKGIGEKTARKLLEEWGSLEALLKNLDRLKPAIREKILAHMDDLKLSWDLAKVRTDLPLEVDFAKRREPDRERLRAFLERLEFGSLLHEFGLLESPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEEIARLEAEVFRLAGHPFNLNSRDQLERVLFDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDLIHPRTGRLHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQIELRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINFGVLYGMSAHRLSQELAIPYEEAQAFIERYFQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWLSAKEGGGGSGGGGSGGGGSMAGYGAKGIRKVGAFRSGLEDKVSKQLESKGIKFEYEEWKVPYVIPASNHTYTPDFLLPNGIFVETKGLWESDDRKKHLLIREQHPELDIRIVFSSSRTKLYKGSPTSYGEFCEKHGIKFADKLIPAEWIKEPKKEVPFDRLKRKGGKKGGGGSGGGGSGGGGSMEGMLKGEGPGPLPPLLQQYVELRDQYPDYLLLFQVGDFYECFGEDAERLARALGLVLTHKTSKDFTTPMAGIPLRAFEAYAERLLKMGFRLAVADQVEPAEEAEGLVRREVTQLLTPGTLLQESLLPREANYLAAIATGDGWGLAFLDVSTGEFKGTVLKSKSALYDELFRHRPAEVLLAPELLENGAFLDEFRKRFPVMLSEAPFEPEGEGPLALRRARGALLAYAQRTQGGALSLQPFRFYDPGAFMRLPEATLRALEVFEPLRGQDTLFSVLDETRTAPGRRLLQSWLRHPLLDRGPLEARLDRVEGFVREGALREGVRRLLYRLADLERLATRLELGRASPKDLGALRRSLQILPELRALLGEEVGLPDLSPLKEELEAALVEDPPLKVSEGGLIREGYDPDLDALRAAHREGVAYFLELEERERERTGIPTLKVGYNAVFGYYLEVTRPYYERVPKEYRPVQTLKDRQRYTLPEMKEKEREVYRLEALIRRREEEVFLEVRERAKRQAEALREAARILAELDVYAALAEVAVRYGYVRPRFGDRLQIRAGRHPVVERRTEFVPNDLEMAHELVLITGPNMAGKSTFLRQTALIALLAQVGSFVPAEEAHLPLFDGIYTRIGASDDLAGGKSTFMVEMEEVALILKEATENSLVLLDEVGRGTSSLDGVAIATAVAEALHERRAYTLFATHYFELTALGLPRLKNLHVAAREEAGGLVFYHQVLPGPASKSYGVEVAAMAGLPKEVVARARALLQAMAARREGALDAVLERLLALDPDRLTPLEALRLLQELKALALGAPLDTMKG
Further, the amino acid sequence of Taq DNA polymerase-linker-ExonucleaseIII-linker-Thermus aquaticus Muts is shown in SEQ ID NO. 2, and is specifically as follows:
MRGMLPLFEPKGRVLLVDGHHLAYRTFHALKGLTTSRGEPVQAVYGFAKSLLKALKEDGDAVIVVFDAKAPSFRHEAYGGYKAGRAPTPEDFPRQLALIKELVDLLGLARLEVPGYEADDVLASLAKKAEKEGYEVRILTADKDLYQLLSDRIHVLHPEGYLITPAWLWEKYGLRPDQWADYRALTGDESDNLPGVKGIGEKTARKLLEEWGSLEALLKNLDRLKPAIREKILAHMDDLKLSWDLAKVRTDLPLEVDFAKRREPDRERLRAFLERLEFGSLLHEFGLLESPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEEIARLEAEVFRLAGHPFNLNSRDQLERVLFDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDLIHPRTGRLHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQIELRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINFGVLYGMSAHRLSQELAIPYEEAQAFIERYFQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWLSAKEGGGGSGGGGSGGGGSMKFVSFNINGLRARPHQLEAIVEKHQPDVIGLQETKVHDDMFPLEEVAKLGYNVFYHGQKGHYGVALLTKETPIAVRRGFPGDDEEAQRRIIMAEIPSLLGNVTVINGYFPQGESRDHPIKFPAKAQFYQNLQNYLETELKRDNPVLIMGDMNISPTDLDIGIGEENRKRWLRTGKCSFLPEEREWMDRLMSWGLVDTFRHANPQTADRFSWFDYRSKGFDDNRGLRIDLLLASQPLAECCVETGIDYEIRSMEKPSDHAPVWATFRRGGGGSGGGGSGGGGSMEGMLKGEGPGPLPPLLQQYVELRDQYPDYLLLFQVGDFYECFGEDAERLARALGLVLTHKTSKDFTTPMAGIPLRAFEAYAERLLKMGFRLAVADQVEPAEEAEGLVRREVTQLLTPGTLLQESLLPREANYLAAIATGDGWGLAFLDVSTGEFKGTVLKSKSALYDELFRHRPAEVLLAPELLENGAFLDEFRKRFPVMLSEAPFEPEGEGPLALRRARGALLAYAQRTQGGALSLQPFRFYDPGAFMRLPEATLRALEVFEPLRGQDTLFSVLDETRTAPGRRLLQSWLRHPLLDRGPLEARLDRVEGFVREGALREGVRRLLYRLADLERLATRLELGRASPKDLGALRRSLQILPELRALLGEEVGLPDLSPLKEELEAALVEDPPLKVSEGGLIREGYDPDLDALRAAHREGVAYFLELEERERERTGIPTLKVGYNAVFGYYLEVTRPYYERVPKEYRPVQTLKDRQRYTLPEMKEKEREVYRLEALIRRREEEVFLEVRERAKRQAEALREAARILAELDVYAALAEVAVRYGYVRPRFGDRLQIRAGRHPVVERRTEFVPNDLEMAHELVLITGPNMAGKSTFLRQTALIALLAQVGSFVPAEEAHLPLFDGIYTRIGASDDLAGGKSTFMVEMEEVALILKEATENSLVLLDEVGRGTSSLDGVAIATAVAEALHERRAYTLFATHYFELTALGLPRLKNLHVAAREEAGGLVFYHQVLPGPASKSYGVEVAAMAGLPKEVVARARALLQAMAARREGALDAVLERLLALDPDRLTPLEALRLLQELKALALGAPLDTMKG
The DNA synthesis error correction enzyme is applied to DNA artificial synthesis.
The invention has the beneficial effects that:
The DNA synthesis error correction enzyme is formed by connecting a structural domain for catalyzing DNA synthesis in Taq DNA polymerase, an error correction active structural domain in enzyme for correcting DNA mismatch and a functional structural domain for specifically recognizing mismatched DNA double chains in MutS protein by using a flexible linker. The DNA synthesis error correction enzyme has three functions of DNA catalytic synthesis, specific recognition and mismatch DNA double-chain repair, so that the base error rate is less than 10 -3, the error correction efficiency and the cloning accuracy are good, the single error correction step in DNA artificial synthesis is omitted, and the efficiency of DNA artificial synthesis is improved.
Detailed Description
The technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
Preparation Taq DNA polymerase-linker-T7 EndonucleaseI-linker-Thermus aquaticus Muts, comprising the following steps:
Searching a DNA sequence of a structural domain for catalyzing DNA synthesis in Taq DNA polymerase, a DNA sequence of a DNA mismatch error correction active structural domain in T7 endonuclease I and a DNA sequence of a functional domain for specifically recognizing DNA double-strand mismatch in MutS protein in PUBMED database, linearly connecting the three by using a flexible linker, respectively introducing enzyme cutting sites NdeI and XhoI at the N end and the C end of the three to synthesize a recombinant sequence 1, inserting the recombinant sequence 1 into an expression vector pET-22b (+) to obtain a recombinant plasmid pET-22b (+) -1, and then converting the recombinant plasmid pET-22b (+) -1 into an escherichia coli cloning strain DH5 alpha.
Step two, selecting DH5 alpha monoclonal strain containing recombinant plasmid pET-22b (+) -1, inoculating to LB culture medium, placing in a shaking table, culturing for 3h under the conditions of 220r/min and 37 ℃ to enable thalli to be in logarithmic growth phase, reducing the temperature of the shaking table to 28 ℃, inducing expression of target protein by using IPTG (isopropyl thio-beta-D-galactoside) with the final concentration of 0.4mmol/L, centrifuging for 20min under the condition of 4000r/min, collecting thalli, then adding lysozyme with the concentration of 300mg/mL, cracking for 30min on ice, performing ultrasonic crushing, centrifuging, collecting supernatant, purifying the supernatant by magnetic beads, eluting and collecting purified protein to obtain Taq DNA polymerase-linker-T7 EndonucleaseI-linker-Thermusaquaticus Muts.
The DNA sequence of recombinant sequence 1 is as follows:
catATGCGTGGTATGCTGCCGCTGTTTGAACCGAAAGGCCGTGTGCTGCTGGTGGATGGTCATCATCTGGCCTATCGCACCTTTCATGCACTGAAAGGCCTGACCACCAGTCGCGGTGAACCGGTTCAGGCAGTTTATGGTTTTGCAAAAAGCCTGCTGAAAGCACTGAAAGAAGATGGTGACGCAGTGATTGTTGTGTTTGATGCAAAAGCCCCGAGTTTTCGTCATGAAGCATACGGTGGCTATAAAGCAGGTCGCGCACCGACCCCGGAAGATTTTCCGCGTCAGCTGGCCCTGATTAAGGAACTGGTGGATCTGCTGGGCCTGGCCCGTCTGGAAGTGCCGGGTTATGAAGCCGATGATGTGCTGGCAAGTCTGGCAAAAAAGGCAGAAAAAGAAGGTTATGAAGTTCGTATTCTGACCGCCGATAAAGATCTGTATCAGCTGCTGAGTGATCGTATTCATGTGCTGCATCCGGAAGGCTATCTGATTACCCCGGCATGGCTGTGGGAAAAATATGGCCTGCGCCCGGATCAGTGGGCCGATTATCGTGCCCTGACCGGCGATGAAAGCGATAATCTGCCGGGTGTTAAAGGTATTGGCGAAAAAACCGCCCGTAAACTGCTGGAAGAATGGGGTAGCCTGGAAGCCCTGCTGAAAAATCTGGATCGCCTGAAACCGGCCATTCGTGAAAAAATTCTGGCCCACATGGATGATCTGAAACTGAGCTGGGATCTGGCCAAAGTTCGTACCGATCTGCCGCTGGAAGTTGATTTTGCAAAACGCCGCGAACCGGATCGTGAACGCCTGCGCGCATTTCTGGAACGCCTGGAATTTGGCAGCCTGCTGCATGAATTTGGTCTGCTGGAAAGCCCGAAAGCCCTGGAAGAAGCCCCGTGGCCGCCGCCTGAAGGCGCATTTGTGGGCTTTGTGCTGAGTCGTAAAGAACCGATGTGGGCCGATCTGCTGGCACTGGCAGCCGCACGTGGTGGTCGTGTTCATCGTGCACCGGAACCGTATAAAGCCCTGCGTGATCTGAAAGAAGCCCGTGGTCTGCTGGCCAAAGATCTGAGTGTGCTGGCCCTGCGTGAAGGCCTGGGTCTGCCGCCTGGCGATGATCCGATGCTGCTGGCCTATCTGCTGGATCCGAGCAATACCACCCCGGAAGGTGTGGCACGCCGTTATGGCGGTGAATGGACCGAAGAAGCCGGTGAACGTGCAGCCCTGAGTGAACGTCTGTTTGCCAATCTGTGGGGCCGCCTGGAAGGTGAAGAACGCCTGCTGTGGCTGTATCGTGAAGTTGAACGTCCGCTGAGTGCCGTTCTGGCACACATGGAAGCAACCGGTGTTCGTCTGGATGTTGCCTATCTGCGTGCCCTGAGTCTGGAAGTTGCCGAAGAAATTGCCCGCCTGGAAGCCGAAGTTTTTCGTCTGGCAGGCCATCCGTTTAATCTGAATAGTCGCGATCAGCTGGAACGCGTTCTGTTTGATGAACTGGGCCTGCCGGCCATTGGCAAAACCGAAAAAACCGGTAAACGTAGCACCAGCGCCGCAGTTCTGGAAGCACTGCGCGAAGCCCATCCGATTGTGGAAAAAATTTTACAGTATCGTGAGCTGACCAAACTGAAAAGCACCTATATTGATCCGCTGCCGGATCTGATTCATCCGCGTACCGGTCGTCTGCATACCCGTTTTAATCAGACCGCAACCGCCACCGGCCGCCTGTCAAGTAGTGATCCGAATCTGCAGAATATTCCGGTTCGTACCCCGCTGGGCCAGCGTATTCGCCGTGCATTCATTGCAGAAGAAGGCTGGCTGCTGGTTGCCCTGGATTATAGTCAGATTGAACTGCGCGTTCTGGCACATCTGAGTGGCGATGAAAATCTGATTCGCGTTTTTCAGGAAGGCCGCGATATTCATACCGAAACCGCAAGCTGGATGTTTGGCGTTCCGCGTGAAGCCGTTGATCCGCTGATGCGCCGTGCAGCCAAAACCATTAATTTTGGCGTGCTGTATGGCATGAGCGCACATCGTCTGAGTCAGGAACTGGCAATTCCGTATGAAGAAGCCCAGGCATTCATTGAACGTTATTTTCAGAGCTTTCCGAAAGTGCGCGCCTGGATTGAAAAAACCCTGGAAGAAGGCCGCCGCCGTGGTTATGTTGAAACCCTGTTTGGTCGCCGCCGTTATGTGCCGGATCTGGAAGCACGCGTGAAAAGCGTTCGTGAAGCAGCCGAACGTATGGCATTCAATATGCCGGTTCAGGGCACCGCAGCAGATCTGATGAAACTGGCAATGGTTAAACTGTTTCCGCGTCTGGAAGAAATGGGCGCCCGTATGCTGCTGCAGGTTCATGATGAACTGGTGCTGGAAGCACCGAAAGAACGTGCAGAAGCCGTGGCCCGTCTGGCCAAAGAAGTTATGGAAGGTGTGTATCCGCTGGCCGTTCCGCTGGAAGTGGAAGTGGGTATTGGTGAAGATTGGCTGAGCGCAAAAGAAGGTGGCGGTGGCAGTGGCGGCGGTGGTTCAGGTGGCGGTGGTAGCATGGCCGGCTATGGTGCCAAAGGTATTCGTAAAGTTGGTGCCTTTCGCAGCGGCCTGGAAGATAAAGTTAGTAAACAGCTGGAAAGCAAAGGTATTAAGTTTGAATATGAGGAATGGAAAGTGCCGTATGTTATTCCGGCCAGTAATCATACCTATACCCCGGATTTTCTGCTGCCGAATGGCATTTTTGTTGAAACCAAAGGCCTGTGGGAAAGTGATGATCGTAAAAAACATCTGCTGATTCGTGAACAGCATCCGGAACTGGATATTCGTATTGTTTTTAGCAGTAGCCGCACCAAACTGTATAAAGGCAGTCCGACCAGCTATGGTGAATTTTGTGAAAAACATGGTATCAAATTCGCAGATAAACTGATTCCGGCAGAATGGATTAAGGAACCGAAAAAAGAAGTGCCGTTTGATCGTCTGAAACGTAAAGGCGGCAAAAAAGGTGGTGGCGGCAGTGGCGGTGGTGGTAGTGGCGGTGGCGGCTCAATGGAAGGTATGCTGAAAGGCGAAGGTCCGGGCCCGCTGCCGCCTTTACTGCAGCAGTATGTGGAACTGCGTGATCAGTATCCGGATTATCTGCTGCTGTTTCAGGTTGGCGATTTTTATGAATGTTTTGGTGAAGATGCCGAACGCCTGGCCCGCGCCCTGGGTTTAGTGCTGACCCATAAAACCAGTAAAGATTTTACCACCCCGATGGCAGGTATTCCGCTGCGCGCCTTTGAAGCCTATGCCGAACGCTTACTGAAAATGGGCTTTCGTCTGGCGGTTGCAGATCAGGTTGAACCGGCAGAAGAAGCAGAAGGTCTGGTTCGTCGTGAAGTGACCCAGCTGCTGACCCCGGGCACCCTGCTGCAGGAAAGCCTGCTGCCGCGCGAAGCCAATTATCTGGCAGCCATTGCAACCGGCGATGGCTGGGGTCTGGCCTTTCTGGATGTTAGTACCGGTGAATTCAAAGGTACCGTGCTGAAAAGCAAAAGTGCCCTGTATGATGAACTGTTTCGTCATCGTCCGGCCGAAGTGCTGCTGGCCCCGGAACTGCTGGAAAATGGCGCATTTCTGGATGAATTTCGTAAACGCTTTCCGGTTATGCTGAGCGAAGCACCGTTTGAACCGGAAGGTGAAGGCCCGCTGGCCCTGAGACGTGCACGTGGTGCCCTGCTGGCCTACGCCCAGCGTACCCAGGGCGGTGCACTGAGTCTGCAGCCGTTTCGTTTTTATGATCCGGGTGCCTTTATGCGTCTGCCGGAAGCCACCCTGCGTGCACTGGAAGTTTTTGAACCGCTGCGCGGTCAGGATACCCTGTTTAGTGTTCTGGATGAAACCCGTACCGCACCGGGTCGTCGTCTGCTGCAGAGCTGGCTGCGTCATCCGCTGCTGGATCGTGGCCCGCTGGAAGCCCGTCTGGATCGTGTTGAAGGTTTTGTGCGCGAAGGTGCCCTGCGTGAGGGTGTTCGCCGCCTGCTGTATCGTCTGGCAGATCTGGAACGCTTAGCAACCCGTCTGGAACTGGGCCGCGCAAGCCCGAAAGATCTGGGTGCACTGCGCCGCAGCCTGCAGATTCTGCCGGAACTGCGTGCATTACTGGGTGAAGAAGTGGGTCTGCCGGATCTTAGTCCGCTGAAAGAAGAACTGGAAGCCGCCCTGGTTGAAGATCCGCCGCTGAAAGTTAGCGAAGGTGGCCTGATTCGTGAGGGTTATGATCCGGATCTGGATGCCCTGCGCGCAGCCCATCGCGAAGGCGTTGCATATTTTCTGGAACTGGAAGAACGTGAACGTGAACGCACCGGCATTCCGACCCTGAAAGTTGGCTATAATGCCGTGTTTGGTTATTATCTGGAAGTGACCCGCCCGTATTATGAACGCGTTCCGAAAGAATATCGTCCGGTTCAGACCCTGAAAGATCGTCAGCGTTATACCCTGCCGGAAATGAAAGAAAAAGAACGTGAAGTTTACCGTCTGGAAGCATTAATTCGCCGTCGCGAAGAAGAAGTGTTTCTGGAAGTTCGCGAACGCGCAAAACGCCAGGCAGAAGCCCTGCGCGAAGCAGCACGCATTCTGGCAGAACTGGATGTTTATGCCGCCCTGGCAGAAGTTGCCGTGCGCTATGGCTATGTTCGTCCGCGTTTTGGCGATCGCCTGCAGATTCGCGCAGGCCGTCATCCGGTGGTTGAACGCCGTACCGAATTTGTGCCGAATGATCTGGAAATGGCCCATGAACTGGTGTTAATTACCGGTCCGAATATGGCAGGCAAAAGTACCTTTCTGCGTCAGACCGCCCTGATTGCCCTGCTGGCACAGGTTGGTAGTTTTGTGCCGGCAGAAGAGGCCCATCTGCCGCTGTTCGATGGTATCTATACCCGCATTGGTGCCAGCGATGATCTGGCAGGCGGTAAAAGCACCTTTATGGTTGAAATGGAAGAAGTGGCACTGATTCTGAAAGAAGCAACCGAAAATAGTCTGGTGCTGCTGGATGAAGTTGGCCGTGGTACCAGTAGCCTGGATGGCGTTGCAATTGCAACCGCAGTGGCAGAAGCCTTACATGAACGCCGCGCCTATACCCTGTTTGCCACCCATTATTTTGAACTGACCGCACTGGGCCTGCCTCGTCTGAAAAATCTTCATGTTGCAGCACGTGAAGAAGCCGGCGGTCTGGTGTTTTATCATCAGGTGCTGCCGGGTCCGGCAAGCAAAAGTTATGGCGTGGAAGTTGCAGCAATGGCCGGCCTGCCGAAAGAAGTGGTTGCCCGCGCACGTGCCCTGCTGCAGGCAATGGCAGCACGTCGCGAAGGCGCCCTGGATGCAGTTCTGGAGCGCCTGCTGGCATTAGATCCGGATCGTCTGACCCCGCTGGAAGCGCTGCGCCTGCTGCAGGAGCTGAAAGCATTAGCACTGGGTGCACCGCTGGATACCATGAAAGGCTAATAActcgag
The amino acid sequence of Taq DNA polymerase-linker-T7 EndonucleaseI-linker-Thermus aquaticus Muts is shown in SEQ ID NO. 2.
Example 2
Preparation Taq DNA polymerase-linker-ExonucleaseIII-linker-Thermus aquaticus Muts, comprising the following steps:
Searching a DNA sequence of a structural domain for catalyzing DNA synthesis in Taq DNA polymerase, a DNA sequence of a DNA mismatch error correction active structural domain in exonuclease III and a DNA sequence of a functional domain for specifically recognizing DNA double-strand mismatch in MutS protein in PUBMED database, linearly connecting the three by using a flexible linker, respectively introducing enzyme cutting sites NdeI and XhoI at the N end and the C end of the three to synthesize a recombinant sequence 2, inserting the recombinant sequence 2 into an expression vector pET-22b (+) to obtain a recombinant plasmid pET-22b (+) -2, and converting the recombinant plasmid pET-22b (+) -2 into an escherichia coli cloning strain DH5 alpha.
Step two, selecting DH5 alpha monoclonal strain containing recombinant plasmid pET-22b (+) -2, inoculating to LB culture medium, placing in a shaking table, culturing for 3h under the conditions of 220r/min and 37 ℃ to enable thalli to be in logarithmic growth phase, reducing the temperature of the shaking table to 28 ℃, inducing expression of target protein by using IPTG (isopropyl thio-beta-D-galactoside) with the final concentration of 0.4mmol/L, centrifuging for 20min under the condition of 4000r/min, collecting thalli, then adding lysozyme with the concentration of 300mg/mL, cracking for 30min on ice, performing ultrasonic crushing, centrifuging, collecting supernatant, purifying the supernatant by magnetic beads, eluting and collecting purified protein to obtain Taq DNA polymerase-linker-ExonucleaseIII-linker-Thermus aquaticus Muts.
The DNA sequence of recombinant sequence 2 is as follows:
catATGCGTGGCATGCTGCCGCTGTTTGAACCGAAAGGCCGTGTTCTGCTGGTTGATGGTCATCATCTGGCATATCGCACCTTTCATGCACTGAAAGGTCTGACCACCAGTCGCGGCGAACCGGTTCAGGCAGTGTATGGCTTTGCAAAAAGCCTGCTGAAAGCACTGAAAGAAGATGGTGACGCAGTGATTGTGGTGTTTGATGCCAAAGCACCGAGTTTTCGTCATGAAGCATACGGTGGTTATAAAGCAGGCCGTGCACCGACCCCGGAAGATTTTCCGCGTCAGCTGGCCCTGATTAAGGAACTGGTGGATCTGCTGGGTCTGGCACGCCTGGAAGTTCCGGGCTATGAAGCAGATGATGTGCTGGCCAGTCTGGCAAAAAAGGCAGAAAAAGAAGGTTATGAAGTTCGTATTCTGACCGCCGATAAAGATCTGTATCAGCTGCTGAGTGATCGTATTCATGTTCTGCATCCGGAAGGCTATCTGATTACCCCGGCCTGGCTGTGGGAAAAATATGGCCTGCGTCCGGATCAGTGGGCAGATTATCGTGCACTGACCGGTGACGAAAGCGATAATCTGCCGGGCGTTAAAGGTATTGGTGAAAAAACCGCCCGTAAACTGCTGGAAGAATGGGGTAGCCTGGAAGCACTGCTGAAAAATCTGGATCGCCTGAAACCGGCAATTCGCGAAAAAATTCTGGCCCACATGGATGATCTGAAACTGAGCTGGGATCTGGCAAAAGTGCGCACCGATCTGCCGCTGGAAGTGGATTTTGCAAAACGCCGTGAACCGGATCGTGAACGCCTGCGCGCCTTTCTGGAACGTCTGGAATTTGGCAGCCTGCTGCATGAATTTGGTCTGCTGGAAAGTCCGAAAGCACTGGAAGAAGCCCCGTGGCCGCCGCCTGAAGGCGCATTTGTTGGCTTTGTGCTGAGTCGCAAAGAACCGATGTGGGCCGATCTGCTGGCACTGGCCGCCGCACGTGGCGGTAGAGTTCATCGCGCACCGGAACCGTATAAAGCACTGCGCGATCTGAAAGAAGCCCGCGGCCTGCTGGCAAAAGATCTGAGCGTTCTGGCACTGCGCGAAGGTCTGGGTCTGCCGCCGGGTGACGATCCGATGCTGCTGGCCTATCTGCTGGATCCGAGCAATACCACCCCGGAAGGTGTGGCACGTCGTTATGGTGGTGAATGGACCGAAGAAGCAGGCGAACGTGCCGCACTGAGCGAACGCCTGTTTGCCAATCTGTGGGGTCGTCTGGAAGGCGAAGAACGCCTGCTGTGGCTGTATCGTGAAGTGGAACGCCCGCTGAGCGCAGTGCTGGCCCACATGGAAGCAACCGGCGTGCGCCTGGATGTGGCCTATCTGCGTGCCCTGAGTCTGGAAGTTGCAGAAGAAATTGCCCGTCTGGAAGCAGAAGTGTTTCGCCTGGCCGGCCATCCGTTTAATCTGAATAGTCGCGATCAGCTGGAACGTGTGCTGTTTGATGAACTGGGCCTGCCGGCAATTGGCAAAACCGAAAAAACCGGTAAACGCAGTACCAGCGCCGCCGTTCTGGAAGCCCTGCGCGAAGCCCATCCGATTGTTGAAAAAATTTTACAGTACCGTGAGCTGACCAAACTGAAAAGTACCTATATTGATCCGCTGCCGGATCTGATTCATCCGCGTACCGGCCGCCTGCATACCCGTTTTAATCAGACCGCAACCGCCACCGGTCGTCTGAGCAGTAGTGATCCGAATCTGCAGAATATTCCGGTGCGCACCCCGCTGGGCCAGCGTATTCGCCGTGCCTTTATTGCCGAAGAAGGTTGGCTGCTGGTGGCCCTGGATTATAGTCAGATTGAACTGCGTGTTCTGGCACATCTGAGCGGTGACGAAAATCTGATTCGTGTGTTTCAGGAAGGTCGCGATATTCATACCGAAACCGCAAGTTGGATGTTTGGCGTTCCGCGCGAAGCAGTTGATCCGCTGATGCGCCGTGCAGCCAAAACCATTAATTTTGGTGTGCTGTATGGCATGAGTGCCCATCGTCTGAGTCAGGAACTGGCAATTCCGTATGAAGAAGCACAGGCCTTTATTGAACGCTATTTTCAGAGCTTTCCGAAAGTTCGTGCATGGATTGAAAAAACCCTGGAAGAAGGTCGTCGCCGTGGTTATGTTGAAACCCTGTTTGGTCGTCGCCGCTATGTTCCGGATCTGGAAGCACGCGTGAAAAGCGTTCGCGAAGCCGCAGAACGTATGGCCTTTAATATGCCGGTTCAGGGTACCGCCGCCGATCTGATGAAACTGGCAATGGTGAAACTGTTTCCGCGCCTGGAAGAAATGGGCGCCCGTATGCTGCTGCAGGTGCATGATGAACTGGTTCTGGAAGCGCCGAAAGAACGTGCCGAAGCCGTTGCCCGTCTGGCAAAAGAAGTTATGGAAGGCGTGTATCCGCTGGCCGTGCCGCTGGAAGTTGAAGTTGGTATTGGTGAGGATTGGCTGAGCGCCAAAGAAGGCGGCGGTGGTAGTGGCGGCGGCGGTAGCGGTGGTGGTGGTTCAATGAAATTTGTGAGCTTTAATATCAACGGTCTGCGTGCACGCCCGCATCAGCTGGAAGCCATTGTTGAAAAGCATCAGCCGGATGTTATTGGTCTGCAGGAAACCAAAGTTCATGATGATATGTTTCCGCTGGAAGAAGTGGCCAAACTGGGTTATAATGTGTTTTATCATGGTCAGAAAGGTCATTATGGCGTGGCCCTGCTGACCAAAGAAACCCCGATTGCCGTGCGCCGTGGTTTTCCGGGTGACGACGAAGAAGCCCAGCGCCGCATTATTATGGCCGAAATTCCGAGCCTGCTGGGCAATGTGACCGTTATTAATGGCTATTTTCCGCAGGGTGAAAGTCGTGATCATCCGATTAAGTTTCCGGCCAAAGCCCAGTTTTATCAGAATCTGCAGAACTATCTGGAAACCGAACTGAAACGCGATAATCCGGTGCTGATTATGGGTGACATGAATATTAGCCCGACCGATCTGGATATTGGTATTGGCGAAGAAAATCGTAAACGTTGGCTGCGCACCGGTAAATGTAGCTTTCTGCCGGAAGAACGTGAATGGATGGATCGCCTGATGAGCTGGGGTCTGGTTGATACCTTTCGCCATGCCAATCCGCAGACCGCAGATCGTTTTAGTTGGTTTGATTATCGCAGTAAAGGTTTTGATGATAATCGCGGTCTGCGCATTGATCTGCTGCTGGCAAGTCAGCCGCTGGCAGAATGCTGTGTTGAAACCGGTATTGATTATGAAATTCGTAGCATGGAAAAGCCGAGTGATCATGCCCCGGTGTGGGCCACCTTTCGCCGCGGTGGTGGTGGCAGCGGTGGTGGCGGTAGTGGCGGTGGTGGTAGCATGGAAGGTATGCTGAAAGGTGAAGGTCCGGGCCCGCTGCCGCCGTTACTGCAACAGTATGTTGAACTGCGCGATCAGTATCCGGATTATCTGCTGCTGTTTCAGGTTGGCGATTTTTATGAATGTTTTGGTGAAGATGCCGAACGCCTGGCACGTGCCCTGGGTCTGGTTCTGACCCATAAAACCAGTAAAGATTTTACCACCCCGATGGCCGGTATTCCGCTGCGCGCATTTGAAGCATACGCTGAACGTCTGCTGAAAATGGGCTTTCGCCTGGCGGTGGCCGATCAGGTGGAACCGGCAGAAGAAGCCGAAGGTCTGGTGCGCCGCGAAGTTACCCAGCTGCTGACCCCGGGTACCCTGCTGCAGGAAAGTCTGCTGCCGCGCGAAGCGAATTATCTGGCCGCCATTGCAACCGGTGACGGCTGGGGTCTGGCCTTTCTGGATGTTAGTACCGGCGAATTCAAAGGTACCGTTCTGAAAAGCAAAAGCGCACTGTATGATGAACTGTTTCGCCATCGCCCGGCAGAAGTTCTGCTGGCCCCGGAACTGCTGGAAAATGGTGCCTTTCTGGACGAATTTCGTAAACGCTTTCCGGTTATGCTGAGTGAAGCCCCGTTTGAACCGGAAGGTGAAGGCCCGCTGGCACTGCGTCGCGCTCGTGGTGCACTGCTGGCCTACGCACAGCGTACCCAGGGTGGTGCACTGAGTCTGCAGCCGTTTCGTTTTTATGATCCGGGTGCATTCATGCGCCTGCCGGAAGCCACCCTGCGTGCACTGGAAGTGTTTGAACCGCTGCGTGGTCAGGATACCCTGTTTAGTGTTCTGGATGAAACCCGTACCGCACCGGGTCGTCGCCTGCTGCAGAGTTGGCTGCGTCATCCGCTGCTGGATCGCGGTCCGCTGGAAGCCCGTCTGGATCGTGTTGAAGGTTTTGTGCGTGAAGGTGCACTGCGCGAGGGTGTGCGTCGCCTGTTATATCGTCTGGCCGATCTGGAACGTTTAGCCACCCGTCTGGAACTGGGTCGTGCCAGTCCGAAAGATCTGGGTGCACTGCGTCGTAGTCTGCAGATTCTGCCGGAACTGCGTGCATTACTGGGCGAAGAAGTGGGCCTGCCGGATCTTAGTCCGCTGAAAGAAGAACTGGAAGCAGCACTGGTGGAAGATCCGCCGCTGAAAGTGAGCGAAGGTGGTCTGATTCGCGAAGGTTATGATCCGGATCTGGATGCACTGCGCGCCGCCCATCGTGAAGGCGTTGCCTATTTTCTGGAACTGGAAGAACGTGAGCGTGAACGCACCGGTATTCCGACCCTGAAAGTTGGCTATAATGCCGTGTTTGGCTATTATCTGGAAGTGACCCGCCCGTATTATGAACGCGTTCCGAAAGAATATCGTCCGGTTCAGACCCTGAAAGATCGTCAGCGCTATACCCTGCCGGAAATGAAAGAAAAAGAACGTGAAGTTTACCGTCTGGAAGCGCTGATTCGCCGTCGCGAAGAAGAAGTTTTTCTGGAAGTTCGCGAACGTGCCAAACGCCAGGCAGAAGCACTGCGCGAAGCAGCACGCATTCTGGCAGAACTGGATGTTTATGCAGCACTGGCAGAAGTGGCAGTTCGCTATGGTTATGTTCGTCCGCGTTTTGGTGACCGCCTGCAGATTCGCGCAGGTCGTCATCCGGTTGTGGAACGTCGCACCGAATTTGTTCCGAATGATCTGGAAATGGCACATGAACTGGTTTTAATTACCGGCCCGAATATGGCCGGTAAAAGCACCTTTCTGCGCCAGACCGCACTGATTGCACTGCTGGCACAGGTTGGTAGCTTTGTTCCGGCCGAAGAAGCACATCTGCCGCTGTTCGATGGTATCTATACCCGTATTGGCGCCAGCGATGATCTGGCCGGTGGTAAAAGCACATTCATGGTGGAAATGGAAGAAGTTGCCCTGATTCTGAAAGAAGCGACCGAAAATAGCCTGGTGCTGCTGGATGAAGTGGGCCGCGGCACCAGCAGTCTGGATGGCGTGGCAATTGCCACCGCCGTTGCCGAAGCCCTGCATGAACGTCGCGCATATACCCTGTTTGCCACCCATTATTTTGAACTGACCGCCCTGGGTTTACCGCGTCTGAAAAATCTTCATGTTGCAGCCCGTGAAGAAGCAGGTGGCCTGGTTTTCTATCATCAGGTGCTGCCGGGTCCGGCAAGCAAAAGCTATGGCGTGGAAGTTGCAGCCATGGCAGGCCTGCCGAAAGAAGTTGTGGCACGCGCCCGTGCACTGCTGCAGGCAATGGCCGCCCGCCGTGAAGGCGCACTGGATGCAGTTCTGGAACGCCTGTTAGCCCTGGATCCGGATCGTCTGACCCCGCTGGAAGCATTACGTCTGCTGCAGGAGCTGAAAGCATTAGCACTGGGCGCACCGCTGGATACCATGAAAGGCTAATAActcgag
The amino acid sequence of Taq DNA polymerase-linker-ExonucleaseIII-linker-Thermus aquaticus Muts is shown in SEQ ID NO. 1.
Example 3
1.4 Oligonucleotides were designed and synthesized for preparing mismatched and perfectly correct templates, and oligonucleotides A1B1 and A2B2 were selected for re-denaturation annealing to form A: A mismatched duplex and T: G mismatched duplex, respectively.
The 6 oligonucleotides are shown in Table 1:
TABLE 1
The denaturation system of the oligonucleotide annealing buffers of A1B1 and A2B2 is shown in Table 2:
TABLE 2
Reagent(s) Dosage of
5 XDNA oligonucleotide annealing buffer 10μL
Nuclease-free water 30μL
Oligonucleotide upper strand (100. Mu.M) 5μL
Oligonucleotide lower strand (100. Mu.M) 5μL
The reaction conditions are 95 ℃ and 2min, the temperature of 95 ℃ is slowly reduced to 25 ℃, the temperature reduction rate is 1 ℃ per 0.8s, and the temperature is 4 ℃ and the reaction is preserved.
2. Taking 250ng mismatched double chains, complementing to 10 mu L by using Binding buffer, carrying out denaturation annealing again, and preserving the mismatched double chains at 98 ℃ in 2min, 4 ℃ in 5min, 37 ℃ in 5min and 4 ℃.
Taking 1 the DNA synthesis error correction enzymes prepared in the example 1 and the example 2 respectively, incubating the DNA synthesis error correction enzymes with double chains containing mismatch sites for 5min respectively, enriching, sucking DNA supernatant for one round of PCR amplification, and the reaction system is shown in the table 3:
TABLE 3 Table 3
3. Separating the target fragment from the PCR product by agarose gel electrophoresis, recovering the target fragment by using a gel recovery kit, sequencing by using a 3730XL gene sequencer, and comparing the sequencing result by using Seqman software by referring to the template sequence after sequencing, wherein no base mismatch is found.
Example 4
A1278 bp viral sequence was selected and synthesized as a template, error correction was performed using the error correction enzymes prepared in example 1 and example 2 as a test set, taq DNA polymerase synthesis and error correction without using error correction enzyme, taq DNA polymerase synthesis and error correction with T7 endonuclease I as a control set, and then amplified, recovered, transformed cloned, sequenced using Seqman software, error types, error base numbers, etc., and sequencing results were as shown in Table 4:
TABLE 4 Table 4
As can be seen from Table 4, the error correction enzymes prepared in examples 1 and 2 have good error correction efficiency, the base error rate is less than 10 -3, and the cloning accuracy can reach 80%.
The application of the error correction enzyme does not need a separate error correction step, thereby increasing the efficiency of DNA artificial synthesis.
It should be noted that in this document, terms such as "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims (2)

1. The DNA synthesis error correction enzyme is characterized in that the amino acid sequence of the DNA synthesis error correction enzyme is shown as SEQ ID NO.1 or SEQ ID NO. 2.
2. Use of a DNA synthesis error correction enzyme according to claim 1 in DNA synthesis.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2175490A1 (en) * 1993-11-01 1995-05-11 Paul L. Modrich Methods of analysis and manipulation of dna utilizing mismatch repair systems
CN107686842A (en) * 2016-08-03 2018-02-13 南京大学 A kind of target polynucleotide edit methods and its application

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US6008031A (en) * 1989-05-12 1999-12-28 Duke University Method of analysis and manipulation of DNA utilizing mismatch repair systems
WO2017090685A1 (en) * 2015-11-27 2017-06-01 国立大学法人九州大学 Dna polymerase variant
CN110863049A (en) * 2019-12-06 2020-03-06 苏州卫生职业技术学院 A probe library, detection method and kit for detecting the effectiveness of DNA mismatch repair pathway

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2175490A1 (en) * 1993-11-01 1995-05-11 Paul L. Modrich Methods of analysis and manipulation of dna utilizing mismatch repair systems
CN107686842A (en) * 2016-08-03 2018-02-13 南京大学 A kind of target polynucleotide edit methods and its application

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