DNA synthesis error correction system and application

Abstract

The invention discloses an error correction system capable of efficiently correcting errors in a DNA synthesis process, the system comprises a mixed enzyme system constructed by CELI enzyme and T7 endonuclease I, all mismatch types generated in the DNA synthesis process are specifically recognized and cleaved, and 3%-5% of PEG6000 is added into the error correction system, so that the cleavage effect is remarkably improved. The error correction system can efficiently and specifically recognize and cleave mispairing types possibly generated in the DNA synthesis process, overcomes cleavage preference of cleavage enzyme, and improves the correct fragment acquisition rate in DNA synthesis.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a DNA synthesis error correction system and application BACKGROUND

[0002] DNA design and synthesis is a key common underlying technology to promote the development of life sciences and related fields. Conventional genetic manipulation techniques can only make limited modifications to existing DNA sequences, while DNA synthesis technology can "write" life information from scratch, thereby enhancing our ability to understand, predict and manipulate life from another perspective. The synthesis and assembly of DNA inevitably introduces errors, and the application of error correction techniques based on mismatch binding or mismatch excision at different stages of DNA synthesis can not only improve the accuracy of DNA synthesis, but also effectively reduce the quality control cost of long fragment DNA synthesis.

[0003] Oligonucleotide synthesis and enzymatic assembly processes inevitably produce various types of errors. Common errors include nucleotide insertion, deletion and substitution. The use of error correction techniques can effectively remove different types of errors and improve the accuracy of the synthesis product. Nucleotide insertion, deletion and substitution errors of double-stranded DNA fragments after complementary pairing mainly manifest as mismatches and bulges, and the removal of these errors is more dependent on DNA enzymatic error correction techniques developed based on DNA repair systems in living organisms. By annealing complementary sequences to expose mismatch signals, enzymes with mismatch binding or mismatch cleavage activity are used to correct DNA double strands, thereby enriching correct sequences.

[0004] Mismatch cleavage enzymes are a group of mismatch-specific endonucleases that recognize DNA double-strand mismatch sites and cut near the mismatch sites. They mainly include single-base mismatch-recognizing endonucleases and single-strand-specific nucleases, which work together with polymerases to hydrolytically cleave error regions using polymerases with exonuclease activity. This method can eliminate single-base level errors while retaining most of the sequence-corrected regions. T4 endonuclease VII, T7 endonuclease I and E. coli endonuclease V can recognize and cleave single-base mismatches, single-nucleotide bulges and other types of errors in double-stranded DNA, effectively reducing errors such as mismatches, deletions and insertions in synthetic gene products. CEFI, a single-strand-specific nuclease, can specifically cleave different types of base mismatches and DNA distortions at neutral pH. The above cleavage enzymes have cutting preferences and low cutting efficiency, which to some extent reduces the correct fragment yield in DNA synthesis. SUMMARY

[0005] To solve the above problems, the present application provides an error correction system for DNA synthesis, which corrects all types of mismatches generated during the DNA synthesis process.

[0006] The synthetic error correction system comprises a buffer reagent and a mixed enzyme system.

[0007] The mixed enzyme comprises two or more of CELI endonuclease, T5 endonuclease I, T4 endonuclease VII or T7 endonuclease I.

[0008] Preferably, the enzyme mixture comprises CELI enzyme and T7 endonuclease I.

[0009] Furthermore, based on the amount of substances, the enzyme mixture comprises 60%-70% of CELI enzyme and 30%-40% of T7 endonuclease.

[0010] The buffer reagent includes a buffer and a stabilizer.

[0011] The buffer comprises 10 mM Tris-HCl (pH 7.5), 2 mM to 10 mM Mg 2+ and 10mM~50mM Na + .

[0012] The stabilizer includes one or more of PEG6000, T4GP32 or SSB. Preferably, the stabilizer includes PEG6000.

[0013] Preferably, the final concentration of PEG6000 is between 3% and 5% (m / v).

[0014] Another aspect of the present application provides a DNA synthesis error correction method, which is accomplished using the error correction system.

[0015] The method comprises the following steps: 1) preparing an error-correcting enzyme; 2) configuring a mixed enzyme error-correcting system; and 3) performing an error-correcting reaction.

[0016] The CELI enzyme is prepared by extracting the CELI enzyme from celery, and the T7 endonuclease I is prepared by a microbial synthesis method.

[0017] Beneficial effects of this application:

[0018] Provided are a hybrid error-correcting enzyme system and its preparation and use methods. By adopting a specific error-correcting system, it can comprehensively identify and cut errors such as single-base mismatches and single nucleotide bulges in double-stranded DNA, effectively reducing errors such as mismatches, deletions and insertions in synthetic gene products and improving cutting efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the identification result of CELI endonuclease electrophoresis gel method.

[0020] Figure 2The error correction results of the CELI endonuclease single enzyme system are shown in Figure 1; 1 is a 50 bp DNA ladder; 2 is a perfect match control fragment; 3 is a T:T mismatch template; 4 is a T:G mismatch template; 5 is a T:C mismatch template; 6 is a C:A mismatch template; 7 is a C:C mismatch template; 8 is an A:A mismatch template; 9 is an A:G mismatch template; 10 is a G:G mismatch template; and 11 is a base-deletion mismatch template.

[0021] Figure 3 The error correction results of T7 endonuclease I are shown in Figure 1, where 1 is a 50 bp DNA ladder; 2 is a perfect match control fragment; 3 is a T:T mismatch template; 4 is a T:G mismatch template; 5 is a T:C mismatch template; 6 is a C:A mismatch template; 7 is a C:C mismatch template; 8 is an A:A mismatch template; 9 is an A:G mismatch template; 10 is a G:G mismatch template; and 11 is a base-deletion mismatch template.

[0022] Figure 4 The error correction results of the mixed enzyme system are shown in Figure 1, where 1 is a 50 bp DNA ladder; 2 is a perfectly matched control fragment; 3 is a T:T mismatch template; 4 is a T:G mismatch template; 5 is a T:C mismatch template; 6 is a C:A mismatch template; 7 is a C:C mismatch template; 8 is an A:A mismatch template; 9 is an A:G mismatch template; 10 is a G:G mismatch template; and 11 is a base-deletion mismatch template.

[0023] Figure 5 The activity test results of the error correction systems of Control Examples 1, 2, and 3 are shown, where 1 is a 50 bp DNA ladder; 2 is an error correction system without PEG6000; 3 is the activity test result of the error correction system of Control Example 1; 4 is the activity test result using Example 6; 5 is the activity test result of the error correction system of Control Example 2; and 6 is the activity test result of the error correction system of Control Example 3. DETAILED DESCRIPTION

[0024] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present application will be exemplarily described below with reference to specific embodiments in conjunction with the accompanying drawings. It should be noted that, in this application, relational terms such as first and second are merely used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations.

[0025] The specific implementation methods of this application include:

[0026] 1. Enzyme Preparation

[0027] A) CELI enzyme preparation

[0028] The CELI enzyme is obtained by extracting the CELI enzyme from celery, specifically, comprising the following steps: 1) weighing celery, removing leaves, adding buffer A, pounding into a homogenate, and adjusting the pH of the filtrate to 8.0; 2) keeping warm at 50° C., cooling, and centrifuging to obtain a supernatant; 3) slowly adding ammonium sulfate powder to the obtained supernatant to a saturation of 25%, and centrifuging again to obtain a supernatant; 4) adding ammonium sulfate powder to the supernatant obtained in step 3) to a saturation of 80%, and centrifuging again to retain a precipitate; 5) separating the supernatant from the supernatant; and 4) The precipitate obtained in step 4 is dissolved in buffer B, dialyzed with Tris buffer for desalting, and then concanavalin A lectin is added and stirred overnight; 6) The supernatant is removed using a suction funnel, and buffer is again added to the precipitate to dissolve the enzyme; 7) The filtrate is filtered and concentrated to 30 ml using an ultrafiltration membrane. The filtrate is then applied to a DEAE-Sepharose FF column (30 mm x 400 mm) and gradient elution is performed using buffer solutions D and E as eluents. One tube of eluate is collected every 8 minutes to obtain the CELI nuclease protein eluate.

[0029] In the above steps, the buffer A was prepared by the following method: Na2SO3 and phenylmethylsulfonyl fluoride were added to 0.1M Tris-HCl buffer at pH 8.0 to a concentration of 0.01M and 1mM, respectively; the buffer B was prepared by the following method: phenylmethylsulfonyl fluoride was added to 0.1M Tris-HCl buffer at pH 8.0 to a concentration of 1mM; the buffer C was prepared by the following method: 50mM Methyl mannoside and Triton X-100 were added to a Tris-HCl buffer to a concentration of 30 mM and 0.01%, respectively; the eluent D was prepared by the following method: methyl mannoside, Triton X-100, and KCl were added to a Tris-HCl buffer at pH 8.0 to a concentration of 10 mM, 0.01%, and 10 mM, respectively; the eluent E was prepared by the following method: methyl mannoside, Triton X-100, and KCl were added to a Tris-HCl buffer at pH 8.0 to a concentration of 10 mM, 0.01%, and 0.5 M, respectively.

[0030] The obtained enzyme solution was identified by gel electrophoresis and its content was determined by Coomassie brilliant blue method.

[0031] B) Preparation of T7 Endonuclease I

[0032] The T7 endonuclease I is a protein with a base sequence of SEQ ID No. 1, and is prepared according to the following method:

[0033] (1) Construction of expression vector

[0034] The gene sequence encoding T7 nuclease I protein recorded in NCBI was constructed between the restriction sites NdeI and XhoI of the expression vector pET-28a(+) to obtain a recombinant plasmid.

[0035] (2) Gene expression

[0036] The enzyme was exogenously expressed and purified in E. coli. Specifically, the E. coli expression type recombinant plasmid pET-28a(+)-T7 was transformed into E. coli to obtain recombinant bacteria. Positive clones were screened using kanamycin-resistant plates, and single clones were picked and cultured in 5 mL of LB liquid medium (Kan+, 100 mg / mL) to an OD600 of 0.6-0.8. The bacterial culture was then transferred to 800 mL of 2YT medium (Kan+, 100 mg / ml) and cultured to an OD600 of 0.6-0.8. IPTG was added to a final concentration of 0.5 mM and induced for 16 h. The culture was collected into a collection bottle and centrifuged at 5500 rpm for 15 min. The supernatant was discarded, and the resulting bacterial pellet was suspended in 35 mL of protein buffer (50 mM Tris-HCl, 2 mM EDTA, 0.1% Triton X-100, pH 7.4), poured into a 50 mL centrifuge tube, and stored in a -80°C refrigerator.

[0037] (3) Protein purification

[0038] 2. Preparation of error correction system

[0039] The endonuclease is added to a buffer reagent and a stabilizer is added: 1) the mixed enzyme system includes adding 60%-70% of CELI enzyme and 30%-40% of T7 nuclease I to the buffer reagent; 2) the single enzyme system includes adding CELI enzyme and T7 nuclease I to the buffer reagent respectively; the buffer reagent includes 10mM Tris-HCl (pH 7.5), 2mM-10mM Mg 2+ and 10mM~50mM Na + The stabilizer is PEG6000 with a final concentration of 3%-5% (m / v).

[0040] 3. Error correction response and result evaluation

[0041] This application designs DNA templates with different mismatch types to simulate possible mismatch types to test the error correction ability of the error correction system for various mismatch types, and after the enzyme cleavage reaction is completed, an agarose gel electrophoresis experiment is performed to detect whether the enzyme cleavage experiment is completed.

[0042] (1) Preparation of mismatch template

[0043] Ten oligonucleotides (100 nt in length) were designed and synthesized to prepare mismatched and completely correct templates, and denaturation annealing formed T:T, T:G, T:C, C:A, A:A, A:G, G:G, single-stranded deletion of one base mismatch and completely matched double-stranded, respectively.

[0044] (2) Error correction response

[0045] The mismatch template was subjected to enzyme digestion experiments using an error correction system and terminated with EDTA at a final concentration of 80 mM.

[0046] (3) Analysis of experimental results

[0047] The correction results were analyzed on a 3% agarose gel using agarose electrophoresis. When the DNA duplex with a mismatched base is cleaved by the enzyme at the mismatch site, two bands with a total length of 100 bp are released, which can be used to determine whether the error correction system has been cleaved and corrected at the mismatch site.

[0048] The equipment involved in the implementation process and their sources are shown in Table 1, and the reagents involved and their sources are shown in Table 2.

[0049] Table 1 Equipment and sources

[0050]

[0051] Example 1 Preparation of CELI enzyme

[0052] (1) Enzyme preparation

[0053] Na2SO3 and phenylmethylsulfonyl fluoride were added to 0.1M Tris-HCl buffer at pH 8.0 to a concentration of 0.01M and 1mM, respectively, to obtain buffer A; phenylmethylsulfonyl fluoride was added to 0.1M Tris-HCl buffer at pH 8.0 to a concentration of 1mM to obtain buffer B; methyl mannoside and TritonX-100 were added to 50mM Tris-HCl buffer at pH 8.0 to a concentration of 30mM and 0.01%, respectively, to obtain buffer C; methyl mannoside, TritonX-100 and KCl were added to Tris-HCl buffer at pH 8.0 to a concentration of 10mM, 0.01% and 10mM, respectively, to obtain eluent D; methyl mannoside, TritonX-100 and KCl were added to Tris-HCl buffer at pH 8.0 to a concentration of 10mM, 0.01% and 0.5M, respectively, to obtain eluent E.

[0054] Weigh 10 kg of commercially available celery (i.e., celery), remove the leaves, add 8 L of buffer A, pound into a homogenate, and extract 74 L of filtrate. Adjust the pH of the filtrate to 8.0 with 1 M Tris solution (7.5 L of Tris solution), then incubate at 50°C for 20 minutes and cool on ice. Centrifuge at 5000 x g for 40 minutes, discard the residue, and collect the supernatant. Slowly add ammonium sulfate powder (approximately 144 g per liter) while stirring to 25% saturation. Centrifuge at 5000 x g for 40 minutes, discard the precipitate, collect the supernatant, and add ammonium sulfate powder (approximately 390 g per liter) while stirring to 80% saturation. Centrifuge at 5000 x g for 40 minutes, discard the supernatant, and retain the precipitate. Dissolve the precipitate in 8 L of buffer B and dialyze against the same Tris buffer for desalination. After desalting, 200 ml of Concanavalin A lectin (activated and equilibrated with buffer B) was added to the desalted solution and stirred overnight at 4°C to allow the enzyme to fully bind to the Concanavalin A lectin. After standing for 1 hour, the supernatant was removed with a suction funnel and 300 ml of buffer C was added (in five steps) to dissolve the enzyme in the buffer. The solution was then filtered. The filtrate was concentrated to 30 ml using an ultrafiltration membrane (molecular weight cutoff 10,000 Daltons) and applied to a DEAE-Sepharose FF column (30 mm x 400 mm) using 500 ml each of buffers D and E as eluents. Gradient elution was performed using a TH-500 gradient mixer (Shanghai Huxi Analytical Instrument Factory) at a flow rate of 0.5 ml / min, with one eluate collected every 8 minutes. Protein purity was assessed by SDS-polyacrylamide gel electrophoresis. The enzyme solution from the peak activity tube with a single protein band was pooled. This eluate was the CELI nuclease protein eluate, yielding a total of 20 ml of enzyme solution.

[0055] (2) Enzyme identification

[0056] ① The obtained enzyme solution was detected by SDS-PAGE, and a protein band was observed at 43 kDa, indicating that the obtained enzyme solution should be CELI nuclease, and the purity reached electrophoresis purity (see Figure 1 ).

[0057] Protein content was determined using the Coomassie Brilliant Blue method. The principle is that Coomassie Brilliant Blue G-250 appears red in its free state and turns cyan upon binding to the hydrophobic region of proteins. The former has a maximum absorption at 465 nm, while the latter at 595 nm. Within a certain protein concentration range (0–100 pg / ml), the absorbance of the protein-pigment complex at 595 nm is proportional to the protein content.

[0058] Example 2 Preparation of T7 Endonuclease I

[0059] The T7 endonuclease I used in the following examples is a protein with the amino acid sequence of SEQ ID No. 1, and was prepared according to the following method.

[0060] (1) Construction of expression vector

[0061] The gene sequence encoding T7 nuclease I protein recorded in NCBI was constructed between the restriction sites NdeI and XhoI of the expression vector pET-28a(+) to obtain a recombinant plasmid named pET-28a(+)-T7.

[0062] (2) Gene expression

[0063] Transform the recombinant E. coli expression plasmid pET-28a(+)-T7 into the host strain E. coli BL21(DE3) to obtain recombinant bacteria. Screen positive clones using kanamycin-resistant plates (Kan+, 100 mg / mL) and culture overnight at 37°C. Pick a single colony and transfer it to 5 mL of LB liquid medium (Kan+, 100 mg / mL) and incubate at 37°C, 220 rpm, until the OD600 reaches 0.6-0.8. Transfer 5 mL of the bacterial solution in LB medium to 800 mL of 2YT medium (Kan+, 100 mg / ml), culture at 37°C and 220 rpm until the OD600 reaches 0.6-0.8, then cool to 16°C, add IPTG to a final concentration of 0.5 mM, and induce expression for 16 h; collect the above culture solution into a collection bottle and centrifuge at 5500 rpm for 15 min; discard the supernatant, suspend the resulting bacterial pellet with 35 ml of protein buffer (50 mM Tris-HCl, 2 mM EDTA, 0.1% Triton X-100, pH 7.4), pour into a 50 mL centrifuge tube, and store in a -80°C refrigerator.

[0064] (3) Protein purification

[0065] Disruption of bacteria: Disrupt the above-obtained bacterial pellet twice using a high-pressure, low-temperature disruptor at 1200 bar and 4°C. Centrifuge at 4°C and 10,000 rpm for 45 min. Collect the pellet and supernatant for sample preparation.

[0066] Purification: The supernatant was filtered through a 0.45 μm microporous filter membrane and purified by nickel affinity chromatography. The specific steps are as follows:

[0067] a. Column equilibration: Before loading the supernatant, wash the column with ddH20 for 2 column volumes, then equilibrate the Ni affinity column with protein buffer for 1 column volume;

[0068] b. Loading: Slowly pass the supernatant through the Ni affinity chromatography column at a flow rate of 0.5 mL / min and repeat once;

[0069] c. Elution of impurities: Use protein buffer to wash one column volume, then use 50 mL of protein buffer containing 50 mM imidazole to elute strongly bound impurities, take the first few drops of flow-through sample, and prepare the sample;

[0070] d. Elution of target protein: Elute the target protein with 20 ml of protein buffer containing 100 mM, 200 mM, or 300 mM imidazole, respectively. Take the first few drops of flow-through sample, prepare the sample, and detect it by 12% SDS-PAGE.

[0071] (4) Concentration and fluid replacement

[0072] The collected target protein was concentrated by centrifugation (4°C, 3400 rpm) in a 50 ml Amicon ultrafiltration tube (30 kDa, Millipore) to 1 mL. 10 mL of protein buffer was added and the mixture was concentrated to 1 mL. This process was repeated once to obtain the purified protein T7 endonuclease I.

[0073] (5) Detection concentration

[0074] The protein concentration after concentration was measured using a Nondrop 2000 micro-spectrophotometer and was 0.5 mg / mL, thus obtaining purified and concentrated T7 endonuclease I.

[0075] Example 3 Preparation of mismatched templates

[0076] Ten oligonucleotides (100 nt in length) were designed and synthesized to prepare mismatched and completely correct templates, and denaturation annealing formed T:T, T:G, T:C, C:A, A:A, A:G, G:G, single-stranded one-base-missing mismatched double strands, and completely matched double strands, respectively.

[0077] The oligonucleotide annealing buffer denaturation system for template preparation is shown in Table 3: Reaction conditions: 95°C, 3 min; slowly cooling from 95°C to 25°C at a cooling rate of 1°C / s; and storing at 4°C.

[0078] Table 3 Oligonucleotide annealing buffer denaturation system

[0079] Reagent Amount Oligonucleotide upper strand (50 μM) 4 μL Oligonucleotide lower strand (50 μM) 4 μL 5X DNA oligonucleotide annealing buffer 4 μL Nuclease-free water 8 μL

[0080] Example 4: Error Correction of Mixed Enzyme Cutting System

[0081] Using the annealed product from Example 3 as a template, 300 ng of the annealed product was incubated in 20 μL of a final system (containing 10 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 20 mM NaCl, and 5% PEG6000), 2 μL of CELI enzyme, and 1 μL of T7 endonuclease I at 40°C for 30 minutes. These reactions were terminated with EDTA at a final concentration of 80 mM and analyzed on a 3% agarose gel.

[0082] Example 5: Error Correction of Mixed Enzyme Cutting System

[0083] Using the annealed product from Example 3 as a template, 300 ng of the annealed product was incubated in 20 μL of a final system (containing 10 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 20 mM NaCl, and 3% PEG6000), 2 μL of CELI enzyme, and 1 μL of T7 endonuclease I at 40°C for 30 minutes. These reactions were terminated with EDTA at a final concentration of 80 mM and analyzed on a 3% agarose gel.

[0084] Control Example 1 CELI enzyme error correction

[0085] Using the annealed product obtained in Example 3 as a template, 300 ng of the annealed product was incubated at 42°C for 20 minutes in a final system of 20 μL (containing 10 mM Tris-HCl (pH 7.5), 10 mM MgCl2, and 50 mM KCl) and 2 μL of the CELI enzyme obtained in Example 1. These reactions were terminated with EDTA at a final concentration of 80 mM and analyzed on a 3% agarose gel. Figure 2 From the analysis of the gel results, we concluded that the CELI enzyme showed specific cleavage activity against T:T, T:C, C:A, C:C, A:A, A:G and one-base deletion mismatches.

[0086] Control Example 2 T7 Endonuclease I Error Correction

[0087] Using the annealed product of Example 3 as a template, 300 ng of the annealed product was incubated in 20 μL of a final system (containing 10 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 50 mM NaCl, 1 mM DTT) and 1 μL of T7 endonuclease I at 37°C for 30 minutes. These reactions were terminated with EDTA at a final concentration of 80 mM and analyzed on a 3% agarose gel.

[0088] Control Example 3: Error Correction of Mixed Enzyme Cutting System

[0089] Using the annealed product from Example 3 as a template, 300 ng of the annealed product was incubated in 20 μL of a final system (containing 10 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 20 mM NaCl, 10% PEG6000), 2 μL of CELI enzyme, and 1 μL of T7 endonuclease I at 40°C for 30 minutes. These reactions were terminated with EDTA at a final concentration of 80 mM and analyzed on a 3% agarose gel.

[0090] Control Example 4: Error Correction of Mixed Enzyme Cutting System

[0091] Using the annealed product from Example 3 as a template, 300 ng of the annealed product was incubated in 20 μL of a final system (containing 10 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 20 mM NaCl, 15% PEG6000), 2 μL of CELI enzyme, and 1 μL of T7 endonuclease I at 40°C for 30 minutes. These reactions were terminated with EDTA at a final concentration of 80 mM and analyzed on a 3% agarose gel.

[0092] Results and Discussion:

[0093] In Example 4 and Control Examples 1 and 2, error correction experiments were performed on the same mismatched template using the enzyme mixture described in this application, CELI enzyme, T7 endonuclease I, and enzyme components used as the error correction system. Figure 4 As shown in the figure, three bands appeared in the gel run for mismatch templates of T:T, T:G, T:C, A:A, A:G and G:G, and two bands appeared in the gel run for mismatch templates of C:A, C:C and one base deletion. It can be seen that the mixed enzyme system showed specific cleavage activity for all mismatch types. Figure 2 As shown in the figure, three bands appeared in the gel when the mismatch templates were T:T, T:C, A:A and A:G, and two bands appeared in the gel when the mismatch templates were C:A, C:C and one base deletion. It can be seen that the CELI enzyme showed specific cleavage activity for T:T, T:C, C:A, C:C, A:A, A:G and one base deletion mismatch; the enzyme digestion results of control example 2 were Figure 3 As shown above, two bands appeared in the gel run results of the T:T, T:G, A:G, G:G and one-base deletion mismatch templates, indicating that T7 endonuclease I exhibited specific cleavage activity for T:T, T:G, A:G, G:G and one-base deletion mismatch templates.

[0094] Examples 4, 5 and Control Examples 3 and 4 are based on the error correction enzyme mixed in Example 4, and the error correction experiments are carried out by changing the concentration of PEG6000. The final concentration of PEG6000 in Example 4 is 5%, the final concentration of PEG6000 in Example 5 is 3%, the final concentration of PEG6000 in Control Example 3 is 10%, and the final concentration of PEG6000 in Control Example 4 is 15%. The enzyme cleavage results are shown in FIG. Figure 5 In the figure, the third group of strips is the running result of Example 5, the fourth group of strips is the running result of Example 4, and the fifth and sixth groups of strips are the running results of Control Examples 3 and 4. By comparison, it can be seen that the third and fourth groups of strips are the most obvious and have higher shearing efficiency.

[0095] In summary, the error correction system provided in this application has good specific cleavage activity for all mismatch types.

Claims

1. A DNA synthesis error correction system, characterized in that: The error correction system comprises a buffer reagent and a mixed enzyme system, and can efficiently correct all mismatch types generated during DNA synthesis.

2. The error correction system according to claim 1, wherein the mixed enzyme system comprises two or more of CELI endonuclease, T5 endonuclease I, T4 endonuclease VII or T7 endonuclease I, preferably, comprises CELI enzyme and T7 endonuclease I.

3. The error correction system according to claim 1 or 2, wherein the mixed enzyme system comprises, by substance amount, 60%-70% of CELI enzyme and 30%-40% of T7 endonuclease. The error correction system according to claim 1 or 2, wherein the buffer reagent comprises a buffer and a stabilizer.

5. The error correction system according to claim 1 or 2, wherein the buffer comprises 10 mM Tris-HCl (pH 7.5), 2 mM to 10 mM Mg2+ and 10 mM to 50 mM Na+.

6. The error correction system according to any one of claims 4 or 5, wherein the stabilizer comprises one or more of PEG6000, T4GP32 or SSB, preferably, the stabilizer comprises PEG6000.

7. The error correction system according to any one of claims 4 or 5, wherein the final concentration of PEG6000 is between 3% and 5% (m / v) by volume.

8. A DNA synthesis error correction method, characterized in that: The error correction reaction is completed using the error correction system described in any one of claims 1 to 7.

9. The error correction method according to claim 8, comprising the following steps: 1). Prepare error-correcting enzyme; 2). Configure the error-correcting system; 3). Error-correcting reaction.

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