CN115820625A - Method and device for removing mismatched DNA, preparation method of device and kit - Google Patents

Abstract

The invention relates to the technical field of biology, in particular to a method and a device for removing mismatched DNA in DNA synthesis, a preparation method of the device, and a kit, wherein the method for removing mismatched DNA in a synthesized DNA sample comprises the following steps: binding the mismatched fusion protein to the mismatched DNA in the DNA sample by adding the mismatched fusion protein to the DNA sample; removing the mismatched DNA and mismatched fusion protein using a spin column comprising amorphous cellulose; wherein, the mismatch fusion protein is obtained by fusion expression of the cellulose binding domain fusion protein and the mismatch binding protein. The method for removing the mismatched DNA in the DNA synthesis provided by the invention has the advantages of rapid error correction process and less use of mismatched binding protein. The method controls the volume of the error correction reaction to 10 mu l, can reduce the material cost, is convenient for the application in the aspect of automation, and can complete the error correction in a short time.

Description

Method and device for removing mismatched DNA, preparation method of device and kit

Technical Field

The invention relates to the technical field of biology, in particular to a method and a device for removing mismatched DNA in DNA synthesis, a preparation method of the device and a kit.

Background

With the development of synthetic biology, the demand for genes from different species has increased rapidly, and the design of new functional proteins has also progressed rapidly. DNA-mediated DNA storage technologies have also attracted considerable attention, and these studies and applications have created a great demand for gene synthesis, leading to an increasing market for gene synthesis.

The current DNA synthesis strategy is: oligonucleotides are synthesized by chemical methods, and then the synthesized oligonucleotides are assembled to obtain DNA of about 1Kb, and if longer DNA is required, further assembly is required. Although the reaction efficiency of each chemical synthesis run of the initial four-step oligonucleotide chemical synthesis method can be as high as 99.5%. However, due to various reasons such as insufficient capping reaction, insufficient purity of reaction reagents, too high humidity of reaction environment, etc., the proportion of non-extended oligonucleotides is increased continuously with the increase of the length of the synthesized oligonucleotides, and some side reactions are accompanied in the synthesis process, so that the synthesized product is doped with truncated oligonucleotide products and other chemical reaction byproducts. Therefore, these oligonucleotides are used as materials to assemble and synthesize the gene DNA containing errors such as insertion, deletion and substitution of bases, which requires much time and expense for sequence determination and correction.

In the current separation method of wrong DNA, the electrophoresis method is complex to operate, the immobilization method is relatively simple but still has complex steps, the condition of the magnetic bead method is harsh, and the above problems limit the development of gene synthesis.

Disclosure of Invention

Based on the method, the device, the preparation method of the device and the kit, the method and the device for removing the mismatched DNA in the DNA synthesis are provided, the wrong DNA in the DNA synthesis can be simply removed, the cost of the DNA synthesis is reduced, the reaction volume is reduced, and preparation is provided for application on automatic equipment in the future.

According to a first aspect of the present invention there is provided a method of removing mismatched DNA in a synthetic DNA sample comprising:

adding a mismatch fusion protein to a DNA sample to bind the mismatch fusion protein to a mismatch DNA in the DNA sample;

removing said mismatched DNA and said mismatched fusion protein using a spin column comprising amorphous cellulose;

wherein the mismatch fusion protein is obtained by fusion expression of the cellulose binding domain fusion protein and the mismatch binding protein.

According to an embodiment of the present invention, the removing the mismatch DNA and the mismatch fusion protein using a spin column including the amorphous cellulose comprises:

removing the mismatched DNA in the DNA sample by adding the DNA sample comprising the cellulose binding domain fusion protein and the mismatched binding protein to the spin column.

According to an embodiment of the present invention, the adding of the cellulose binding domain fusion protein and the mismatch binding protein to the DNA sample further comprises: the cellulose binding domain fusion protein and the mismatch binding protein are mixed with the DNA sample, and the mixture is allowed to stand at room temperature until the mismatch DNA binds to the mismatch fusion protein.

According to an embodiment of the present invention, the method further includes: the DNA sample containing only the correct DNA is run out of the column by transient centrifugation.

According to the embodiment of the present invention, the mismatched DNA is obtained by denaturing and annealing DNA in which an incorrect base is synthesized in the DNA sample, and the mismatched DNA mismatched with the correct DNA is obtained.

According to an embodiment of the present invention, the sequence of the fusion protein of the mismatch binding protein and the cellulose binding domain is as shown in SEQ ID NO: as shown at 24.

According to a second aspect of the present invention, there is provided an apparatus for removing mismatched DNA in DNA synthesis for use in the above method, comprising a centrifugal column constructed from a filter element soaked with the above amorphous cellulose and a tip of a pipette, the centrifugal column being used for trapping the mismatched fusion protein to separate the mismatched DNA from the correct DNA in the DNA sample.

According to a third aspect of the present invention, there is provided a method of making the apparatus described above, comprising:

preparing the amorphous cellulose;

and filling the amorphous cellulose into the gun head with the filter element to construct the centrifugal column.

According to an embodiment of the present invention, the above preparing the amorphous cellulose comprises:

dissolving powdered microcrystalline cellulose in phosphoric acid;

precipitating cellulose dissolved in the phosphoric acid;

the cellulose is washed with water and alkali until it becomes neutral, and becomes amorphous cellulose.

According to a fourth aspect of the present invention, there is provided a kit comprising the above-mentioned apparatus for removing mismatched DNA in DNA synthesis.

According to the technical scheme, the method, the device, the preparation method of the device and the kit for removing the mismatched DNA in the DNA synthesis have the following beneficial effects:

the method for removing the mismatched DNA in the DNA synthesis provided by the invention has the advantages that the amorphous cellulose prepared from the microcrystalline cellulose is low in price, and the cost of each error correction reaction is low.

The invention utilizes the characteristic that the regenerated amorphous cellulose can be combined with the cellulose binding domain fusion protein, and utilizes the cheap material-pipette tip with a filter element to construct a device for removing errors, thereby achieving the purpose of removing the wrong DNA. Due to the great simplicity, the device of the invention can be pre-constructed or temporarily constructed when it is necessary to remove faulty DNA. The invention establishes a device and a method for quickly removing DNA errors with high efficiency and convenient operation.

The method for removing the mismatched DNA in the DNA synthesis provided by the invention has the advantages of rapid error correction process and less use of mismatched binding protein. The method controls the volume of the error correction reaction to 10 mu l, can reduce the material cost, is convenient for the application in the aspect of automation, and can complete the error correction in a short time.

Drawings

FIG. 1 is a schematic diagram of an apparatus and a process for rapidly removing DNA with errors according to an embodiment of the present invention;

FIG. 2 is a SDS-PAGE analysis of the EcoMutS fusion protein purified in the examples of the present invention;

FIG. 3 shows the results of the EcoRmutS fusion proteins binding to mismatched DNA in the examples of the present invention;

FIG. 4 shows the result of removing mismatched DNA by an amorphous cellulose spin column in the example of the present invention.

Detailed Description

In order that the objects, technical solutions and advantages of the present invention will become more apparent, the present invention will be further described in detail with reference to the accompanying drawings in conjunction with the following specific embodiments.

At present, there are two main methods for removing errors in DNA synthesis, namely, nuclease cleavage and mismatch binding protein (MutS) binding. After the DNA is denatured and annealed, mismatched DNA is formed between the DNA with errors and the DNA without errors and different types of errors, and perfect pairing is formed between the DNAs without errors. Nuclease in the nuclease cutting method can recognize mismatched bases, cut one strand of the mismatched bases and cut off the mismatched bases, thereby removing errors in DNA synthesis. The mismatch binding protein binding rule is that firstly mutS is bound to mismatched DNA and the complex of mutS and mismatched DNA is removed, leaving only error-free DNA.

At present, the process of the nuclease digestion method is complex, and two enzymes are required to participate. The mismatch binding protein method only requires that the mismatch binding protein MutS recognizes and binds to DNA containing a mismatch, and then separates its complex from the error-free DNA. The electrophoresis method in the separation method is complex to operate, and the immobilization method is relatively simple. The streptavidin involved in the magnetic bead method is expensive, and the preparation of the magnetic beads requires high temperature and high pressure, and the conditions are harsh. Therefore, a simpler and faster method is needed to remove mismatched DNA from a synthetic DNA sample.

According to a first aspect of the present general inventive concept, there is provided a method of removing mismatched DNA in a synthetic DNA sample, comprising:

binding the mismatched fusion protein to the mismatched DNA in the DNA sample by adding the mismatched fusion protein to the DNA sample;

removing the mismatched DNA and mismatched fusion protein using a spin column comprising amorphous cellulose;

wherein, the mismatch fusion protein is obtained by fusion expression of the cellulose binding domain fusion protein and the mismatch binding protein.

According to an embodiment of the invention, the mismatch fusion protein is expressed by fusion of the mismatch binding protein MutS (EcoMutS) of E.coli with the cellulose binding domain 3 (CBM 3). And the DNA sample needing error correction changes the synthesized wrong base into a mismatched base through repeated denaturation annealing; the capability of binding to double-stranded DNA containing mismatched bases can be recognized by the mismatch binding protein MutS, and the double-stranded DNA containing mismatched bases can be bound to MutS.

The invention utilizes the characteristic that the regenerated amorphous cellulose can be combined with the cellulose binding domain fusion protein, and utilizes the cheap material-pipette tip with a filter element to construct a device for removing errors, thereby achieving the purpose of removing the wrong DNA. Due to its great simplicity, the device of the invention can be pre-constructed or temporarily constructed when it is desired to remove faulty DNA. The invention establishes a device and a method for quickly removing DNA errors with high efficiency and convenient operation.

The method for removing the mismatched DNA in the DNA synthesis provided by the invention has the advantages of rapid error correction process and less use of mismatched binding protein. The method controls the volume of the error correction reaction to 10 mu l, can reduce the material cost, is convenient for the application in the aspect of automation, and can complete the error correction in a short time.

In the present invention, "mismatch" refers to missynthesis such as deletion, insertion, and substitution of bases occurring in DNA synthesis. In the present invention, the "fusion protein of EcoMutS and cellulose binding domain" may be simply referred to as "mismatch fusion protein" or "EcoMutS fusion protein".

According to an embodiment of the invention, the use of a spin column comprising amorphous cellulose for the removal of mismatched DNA and mismatched fusion proteins comprises:

mismatched DNA in a DNA sample is removed by adding the DNA sample comprising the cellulose binding domain fusion protein and the mismatched binding protein to a spin column.

According to an embodiment of the invention, wherein the adding of the cellulose binding domain fusion protein and the mismatch binding protein to the DNA sample further comprises: the cellulose binding domain fusion protein and the mismatch binding protein are fully mixed with the DNA sample and are placed at normal temperature until the mismatch DNA is combined with the mismatch fusion protein.

In the examples of the present invention, mismatched DNA is obtained by denaturing and annealing DNA whose base is synthesized as an incorrect base in a DNA sample, and mismatched DNA with the correct DNA.

For example, 18 oligonucleotides for synthesizing Cas12F1 are assembled to synthesize Cas12F1 gene DNA, and started at 100 ℃, slowly cooled, annealed, and mismatches are formed between DNA with errors and between DNA with and without errors.

According to an embodiment of the present invention, the method of removing mismatched DNA in DNA synthesis further comprises: DNA samples containing only the correct DNA were run out of the spin column by transient centrifugation.

The fusion protein solution combined with the mismatched DNA is filled into a gun head with amorphous cellulose, instantaneous centrifugation (spin) is carried out, the mismatched fusion protein of double-stranded DNA with mismatched base is combined by a small column (spin column) of amorphous cellulose and is trapped in the small column, and correct DNA is centrifuged to penetrate through a filter element and the gun head, so that the mismatched DNA is removed, and a correct DNA sample is obtained.

For example, cas12F1 gene DNA assembled is mixed and combined with mismatched DNA, then an amorphous cellulose spin column is added, the mismatched fusion protein and combined DNA are retained by centrifugation, the centrifugation effluent is amplified by PCR and ligated to T vector, escherichia coli DH5a is transformed, clones are randomly selected and sequenced, cas12F1 gene DNA sequences are aligned and error rate is analyzed, and Cas12F1 DNA sequences without error correction are found as a control, and the error rate is reduced from 5.27 parts per million to 0.61 parts per million.

According to an embodiment of the present invention, wherein the sequence of the fusion protein of mismatch binding protein and cellulose binding domain is as shown in SEQ ID NO: as shown at 24.

FIG. 1 is a schematic diagram of an apparatus and a process for rapidly removing erroneous DNA according to an embodiment of the present invention.

According to a second aspect of the present general inventive concept, as shown in fig. 1, there is provided an apparatus for removing mismatched DNA in DNA synthesis according to the method of the present invention, comprising a centrifugal column constructed from a filter element soaked with amorphous cellulose and a tip of a pipette, the centrifugal column being used to retain mismatched fusion protein to separate mismatched DNA from correct DNA in a DNA sample.

According to an embodiment of the invention, the tip of the pipette may be a tip of a 10 μ l pipette

The invention combines the amorphous cellulose column and the mismatch binding protein to establish a device and a method for efficiently removing the error DNA in DNA synthesis. The instantaneous centrifugal column is simple to prepare, low in material cost, quick, simple and convenient in combination of mismatching binding protein, simple in operation process, small in sample volume and small in quantity, and is quick and effective in removing wrong DNA.

According to a third aspect of the present general inventive concept, there is provided a method of manufacturing a device, including:

preparing amorphous cellulose;

amorphous cellulose was packed into a tip with a filter element to construct a centrifugal column.

According to an embodiment of the present invention, wherein preparing amorphous cellulose comprises:

dissolving powdered microcrystalline cellulose in phosphoric acid;

separating out the cellulose dissolved in the phosphoric acid;

the cellulose is washed with water and alkali until neutral, becoming amorphous cellulose.

According to an embodiment of the invention, the amorphous cellulose is stored at 4 ℃.

According to a fourth aspect of the present general inventive concept, there is provided a kit including the apparatus for removing mismatched DNA in DNA synthesis.

The invention combines the amorphous cellulose column and the mismatching binding protein to establish a device and a method for efficiently removing the mismatching DNA in DNA synthesis. The instant centrifugal column has the advantages of simple preparation, low material cost, quick and simple combination of mismatched binding protein, simple operation process, small volume of used samples, and quick and effective removal of the DNA with errors.

The technical solutions of the present invention are described in detail below by using preferred embodiments, and it should be noted that the following specific embodiments are only examples and are not intended to limit the present invention.

Materials and reagents:

all reagents in the present invention are commercially available reagents.

Microcrystalline cellulose Avicel PH105 was purchased from FMC Co (philiadelphia, PA);

coli (Escherichia coli) BL21 (DE 3) is purchased from the family of the engine for the expression of recombinant proteins;

Luria-Bertani (LB) medium containing 1. Mu. Mol/L isopropyl-. Beta. -D-thiogalactoside (IPTG) and containing 100. Mu.g/mL ampicillin was used as the expression medium;

the primers and oligonucleotides were synthesized by Biotechnology engineering (Shanghai) GmbH.

Example 1: sample preparation

(1) Preparing amorphous cellulose gel.

0.2g of microcrystalline cellulose is added into a 50mL centrifuge tube;

adding 0.6mL of deionized water into the cellulose to form a suspension;

adding 10mL of 86.2% H ₃ PO ₄ Slowly stirring;

the cellulose mixture became transparent within a few minutes;

the clear liquid was left at room temperature (or ice bath) for one hour, often vortexed;

about 40mL of ice-cold distilled water was added to slowly precipitate the cellulose (avoiding rapid addition of distilled water) and mixed well.

Centrifuging to remove the supernatant;

washing twice with 40mL of distilled water;

with 2M Na ₂ CO ₃ The cellulose pH was adjusted to about 6 to give amorphous cellulose gel.

(2) Preparation of mismatch binding protein.

FIG. 2 SDS-PAGE analysis of purified EcoMutS fusion proteins.

Coli BL21 (DE 3) cells containing the plasmid pEcoMutS-CBM were inoculated into LB liquid medium containing 100. Mu.g/ml Amp, respectively, and cultured at 37 ℃ before 250rpm until OD was 0.6 to 0.8. Then IPTG was added to a final concentration of 1. Mu.M to induce expression of the target protein. After 16 hours of induction of expression at 16 ℃ the cells were recovered by centrifugation (8000 Xg, 10min, 4 ℃) for purification.

The cells recovered by centrifugation were resuspended in cell disruption buffer (20 mM Tris-HCl buffer (pH 7.6), 300mM KCl,5mM imidazole) and disrupted by ultrasonication (sonic Ultra-cell VCX 130) at 40% strength for 10 minutes for 1 second and 2 seconds. Centrifuging (14000 Xg, 15 min, 4 ℃) to collect the supernatant, and purifying the target protein by using a Ni column; the supernatant was first applied to a Ni column, and since the C-terminus of the protein of interest contained a 6 XHis purification tag, it could bind to the Ni column, followed by washing with a washing buffer (20 mM Tris-HCl buffer (pH 7.6), 300mM KCl,40mM imidazole) to wash away non-specifically bound proteins, and finally elution with an elution buffer (20 mM Tris-HCl buffer (pH 7.6), 300mM KCl,250mM imidazole), and dialysis of the displacement buffer system at 4 ℃. The protein concentration of the target protein was measured using a Bradford protein concentration measurement kit, and bovine serum albumin BSA was used as a standard protein.

FIG. 2 shows the SDS-PAGE analysis of the purified target protein, and it can be seen that MutS is highly pure, but has a small degradation band. From 200mL of cultured E.coli cells, 4mL of the target protein with a concentration of about 5. Mu.M and a purity of more than 90% can be obtained by purification.

Example 2: mismatch binding protein (EcoMutS fusion protein) binding to amorphous cellulose removes mismatch DNA.

(1) Functional verification of the EcoMutS fusion protein.

FIG. 3 results of the EcoRutS fusion protein binding to mismatched DNA.

Mu.l of 10 × annealing buffer (100 mN Tris-HCl, pH 7.6, 500mM NaCl,10mM EDTA), 10. Mu.l of oligonucleotide 1 (10. Mu.M, SEQ ID NO: 1) and 10. Mu.l of oligonucleotide 2 (10. Mu.M, SEQ ID NO: 2) were mixed, denatured at 95 ℃ and slowly annealed to form double-stranded DNA (59 bp) with adenine deletion mismatch (-/T). Oligonucleotide 3 (SEQ ID NO: 3) and oligonucleotide 4 (SEQ ID NO: 4) were similarly annealed to form a double-stranded DNA (54 bp) without mismatch. Then, 0.5. Mu.l each of the annealed double-stranded DNA, 4.5. Mu.l of 5. Mu.M EcoMutS fusion protein, 1. Mu.l of 10 Xbinding buffer (20 mM Tris-HCl, pH 7.6, 100mM KCl,5mM MgCl2) was taken, 3.5. Mu.l of water was added thereto, mixed, allowed to stand at room temperature for 10 minutes, and electrophoresed through 6% PAGE gel.

The results are shown in FIG. 3, in which FIG. 3 (M) shows DNA molecular weight standards;

FIG. 3 (1) shows 59 and 54bp DNAs, in which the 59bp DNA has T/-mismatch;

FIG. 3 (2) shows that the EcoMutS fusion protein binds to mismatched DNA (lane 2 in FIG. 3, no 59bp mismatched protein), indicating that the protein functions normally.

(2) The EcoMutS fusion protein and the bound mismatched DNA were removed by amorphous cellulose.

FIG. 4 results of mismatch DNA removal by amorphous cellulose spin column.

Mu.l of amorphous cellulose (20 mg/ml) was added to 10. Mu.l of a tip with a filter element, and the solution of the EcoMutS fusion protein obtained above binding to mismatched DNA was added to the tip with amorphous cellulose, and after standing for 10min, the solution was subjected to instantaneous centrifugation, the liquid after centrifugation was collected, and after heating at 60 ℃ for 20min, 6-PAGE was carried out. Since the addition of the EcoMutS fusion protein binds to mismatched DNA, only error-free DNA can be seen by PAGE even without removal of the EcoMutS fusion protein, but mismatched DNA remains in solution with the EcoMutS fusion protein, the EcoMutS fusion protein is thermolabile, if not removed, loses the ability to bind to mismatched DNA after heating, and if 59bp of DNA is not detected by PAGE after heating, the removal of the EcoMutS fusion protein together with mismatched DNA is indicated.

Wherein FIG. 4 (M) shows DNA molecular weight standards;

FIG. 4 (1) shows 59 and 54bp DNAs, in which the 59bp DNA has T/-mismatch;

FIG. 4 (2) shows that the EcoMutS fusion protein binds to mismatched DNA;

fig. 4 (3) shows the sample passed through an amorphous cellulose spin column and heated. Heating denatures the EcoMutS fusion protein in the sample and no significant amount of mismatched DNA is detected, indicating that the EcoMutS fusion protein and mismatched DNA are removed together by the amorphous cellulose column.

The liquid collected by centrifugation was subjected to electrophoresis, and it was found that mismatched DNA was greatly reduced in the supernatant using the EcoMutS fusion protein, as shown in FIG. 4 (3), while mismatched DNA (54 bp) remained, indicating that the device of the present invention was capable of removing mismatched DNA.

Example 3: amorphous cellulose and EcoMutS fusion proteins were combined for gene synthesis exemplified by the Cas12F1 gene.

(1) Assembly of Cas12F1 gene DNA

The DNA sequence of the synthesized Cas12F1 gene is shown in SEQ ID NO:5, the DNA sequence of Cas12F1 is split into 18 oligonucleotides (SEQ ID NO:6- -SEQ ID NO: 23) by the software DNAworks, and the assembly of the Cas12F1 gene DNA is performed by Polymerase Cycling Assembly (PCA). The oligonucleotides used in the invention are desalted oligonucleotides synthesized from Shanghai, and the polymerase cycle assembly method comprises the following specific steps:

the first step is as follows: overlapping PCR

The components of the 50. Mu.l reaction system were as follows:

polymerase cycle assembly reaction conditions:

firstly, adjusting the temperature to 94 ℃ for 5 minutes; then maintaining the temperature at 94 ℃ for 15 seconds; the temperature is reduced to 68 ℃ for 30 seconds; then repeatedly adjusting the temperature at 94 ℃ and 68 ℃ and circulating for 25 times; after cycling, the temperature was adjusted to 68 ℃ for 10 minutes, assembly was complete, the polymerase was brought to 4 ℃ and allowed to stand.

The second step is that: the reaction system of PCR is as follows:

oligonucleotide P1 (SEQ ID NO: 1) (10. Mu.M)	2μl
		Oligonucleotide P18 (SEQ ID NO: 18) (10. Mu.M)	2μl
The product obtained in the last PCR step	1μl
		dNTP(2.5mM)	10μl
25mMMgSO4	8μl
		10 XKOD buffer	10μl
KODplusDNA polymerase	2μl
		H 2 O	65μl
Total of	100μl

And (3) PCR reaction conditions:

the temperature of the reactants was first adjusted to 94 ℃ for 5 minutes; then maintaining the temperature at 94 ℃ for 15 seconds; the temperature is reduced to 55 ℃ for 30 seconds; then reducing the temperature to 68 ℃, keeping for 30 seconds, repeatedly adjusting the temperature at 55 ℃ and 68 ℃, and circulating for 30 times; after cycling, the temperature was adjusted to 68 ℃ for 10 minutes, the PCR reaction was completed, and the reaction mixture was placed at 4 ℃ and allowed to stand.

(2) Removal of erroneous DNA

Assembling the Cas12F1 gene obtained above into a product of the second step, performing agarose electrophoresis, performing gel cutting recovery by using a gel recovery kit, performing heating denaturation and renaturation on a certain amount, and removing wrong DNA by using an amorphous cellulose filter element gun head method (spin column). The method comprises the following specific steps:

and (3) denaturation annealing:

mu.L of denaturation buffer (10 Xdenaturation annealing buffer: 10mM Tris-Cl (pH 7.6), 50mM NaCl,1mM EDTA) was added to a 1.5mL centrifuge tube, and DNA (Cas 12F 1) was added to a final concentration of 0.1. Mu. Mol, followed by addition of water to a total volume of 50. Mu.L. Water bath at 95 deg.c for 5 min and slow cooling to room temperature.

Wrong DNA removal:

adding 1 μ l of the obtained solution into a 1.5mL centrifuge tube, adding the EcoMutS fusion protein with the final concentration of 5 μ M, and uniformly mixing at normal temperature for 10 minutes; and simultaneously adding 20 mu l of 20mg/mL amorphous cellulose into a gun head with a filter element, placing the gun head into a 1.5mL centrifuge tube, adding the mixed solution of the EcoMutS fusion protein and the DNA after instantaneous centrifugation, standing the mixture at normal temperature for 10min, and taking the obtained liquid after instantaneous centrifugation as the error-removed DNA.

(3) Determination and analysis of the sequence of the synthesized Cas12F1 gene.

The DNA with the error DNA removed separated by spin column is subjected to assay analysis, and the specific steps are as follows S1-S3:

s1-removal of erroneous DNA amplification:

oligonucleotide P1 (SEQ ID NO: 1) (10. Mu.M)	2μl
		Oligonucleotide P18 (SEQ ID NO: 18) (10. Mu.M)	2μl
Removing fragments after an error	1μl
		dNTP(2.5mM)	10μl
25mMMgSO4	8μl
		10 XKOD buffer	10μl
KODplusDNA polymerase	2μl
		H 2 O	65μl
Total of	100μl

And (3) PCR reaction conditions:

the temperature of the reactants was first adjusted to 94 ℃ for 5 minutes; then maintaining the temperature at 94 ℃ for 15 seconds; the temperature is reduced to 55 ℃ for 30 seconds; then reducing the temperature to 68 ℃, keeping for 30 seconds, repeatedly adjusting the temperature at 55 ℃ and 68 ℃, and circulating for 30 times; after cycling, the temperature was adjusted to 68 ℃ for 10 minutes, the PCR reaction was completed, and the reaction mixture was placed at 4 ℃ and allowed to stand.

S2 construction of vectors

Cas12F1 with errors removed after amplification was ligated to T-vector for about 12 hours at 4 ℃.

S3, vector transformation of Escherichia coli

The above ligation products were mixed with E.coli DH5a competence, transformed as described, spread on LB plates containing 100. Mu.g/mL ampicillin, and cultured overnight at 37 ℃.

Single colonies were randomly picked on the plate and sequenced. The sequencing result is compared with the theoretical sequence of the Cas12F1 gene to be synthesized, and the error rate of Cas12F1 with or without error correction is analyzed. The comparison of error rates in Table 1 shows that the error rate is reduced from 5.27 parts per million to 0.61 parts per million by the method of the present invention.

Table 1 comparison of error rates in synthetic Cas12 gene DNA with and without error correction

By utilizing the device and the method, the test of 59bp double-stranded DNA containing mismatching is firstly carried out, and the test proves that the device and the method are effective; on the basis, the test is carried out by synthesizing the Cas12F1 gene, and the method is proved to be effective, so that the error rate is reduced from 5.27 parts per million to 0.61 part per million. The invention combines the amorphous cellulose column and the mismatching binding protein to establish a device and a method for efficiently removing the mismatching DNA in DNA synthesis. The instant centrifugal column has the advantages of simple preparation, low material cost, quick and simple combination of mismatched binding protein, simple operation process, small volume of used samples, and quick and effective removal of the DNA with errors.

The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are only exemplary embodiments of the present invention and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (10)

1. A method for removing mismatched DNA from a synthetic DNA sample comprising:

binding a mismatched fusion protein to mismatched DNA in a DNA sample by adding the mismatched fusion protein to the DNA sample;

removing the mismatched DNA and the mismatched fusion protein using a spin column comprising amorphous cellulose;

wherein the mismatch fusion protein is obtained by carrying out fusion expression on the cellulose binding domain fusion protein and the mismatch binding protein.

2. The method of claim 1, the removing the mismatch DNA and the mismatch fusion protein using a spin column comprising the amorphous cellulose, comprising:

removing the mismatched DNA in the DNA sample by adding the DNA sample comprising the cellulose binding domain fusion protein and the mismatched binding protein to the spin column.

3. The method of claim 2, wherein said adding said cellulose binding domain fusion protein and said mismatch binding protein to said DNA sample further comprises: and fully mixing the cellulose binding domain fusion protein and the mismatch binding protein with the DNA sample, and placing the mixture at normal temperature until the mismatch DNA is combined with the mismatch fusion protein.

4. The method of claim 2, further comprising:

the DNA sample containing only the correct DNA is run off the spin column by transient centrifugation.

5. The method of claim 4, wherein,

the mismatched DNA is obtained by performing denaturation annealing on DNA with a synthetic incorrect base in the DNA sample, and the mismatched DNA mismatched with the correct DNA is obtained.

6. The method of claim 1, wherein the sequence of the fusion protein of the mismatch binding protein and the cellulose binding domain is as set forth in SEQ ID NO: as shown at 24.

7. An apparatus for removing mismatched DNA in DNA synthesis according to any one of claims 1 to 6, comprising a centrifugal column constructed from a filter element soaked with the amorphous cellulose and a tip of a pipette gun, for trapping the mismatched fusion protein to separate the mismatched DNA from the correct DNA in the DNA sample.

8. A method of making the device of claim 7, comprising:

preparing the amorphous cellulose;

and filling the amorphous cellulose into the gun head with the filter element to construct the centrifugal column.

9. The method for preparing a device for removing mismatched DNA in a synthetic DNA sample according to claim 8, wherein said preparing said amorphous cellulose comprises:

dissolving powdered microcrystalline cellulose in phosphoric acid;

precipitating the dissolved cellulose in the phosphoric acid;

the cellulose is washed with water and alkali until neutral, becoming amorphous cellulose.

10. A kit comprising the apparatus for removing mismatched DNA in DNA synthesis according to claim 7.

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