Preparation method and application of double-strand specific nuclease

Abstract

The invention provides a preparation method and application of double-strand specific nuclease. The method for degrading the DNA in the double-stranded nucleic acid comprises the step of carrying out degradation reaction on the DNA in the double-stranded nucleic acid by utilizing double-stranded specific nuclease, and the double-stranded nucleic acid comprises at least one DNA chain. The double-strand specific nuclease provided by the invention can be used for degrading DNA (Deoxyribonucleic Acid) in double-strand nucleic acid, has higher thermal stability, can be used for better industrial production, and meets the market demand.

Description

[0001] This application is based on the Chinese application with CN application number 202311357856.X and filing date October 18, 2023, and claims its priority. The disclosed content of the CN application is again introduced as a whole into this application. Technical Field

[0002] The present invention relates to the field of enzyme technology, and in particular to a preparation method and application of a double-stranded specific nuclease. Background Art

[0003] DSN, the full name of Duplex-Specific Nuclease, is a thermostable nuclease that was originally isolated from the hepatopancreas of the red king crab (scientific name: Kamchatka crab). It has a molecular weight of 41.5kDa and an isoelectric point of 4.2. The enzyme can selectively degrade double-stranded DNA and DNA in DNA-RNA hybrids, but does not degrade single-stranded nucleic acid molecules. It is widely used in cDNA library construction, SNP detection, and multiple fluorescence detection of miRNA. DSN can accurately distinguish single nucleotide mismatches of 10-12bp DNA-DNA double strands, and DSN can only produce cutting activity when dsDNA is greater than 10bp and DNA-RNA hybrids are greater than 15bp.

[0004] Currently, there is only one company on the market that provides commercial DSN enzyme (Duplex-specific, evrogen, EA003) (lyophilized powder) extracted from the pancreatic tissue of Kamchatka crabs. This commercial enzyme is currently the only heat-resistant DSN in the industry. This commercial enzyme is expressed in inclusion bodies, and the expressed protein needs to be renatured during production. During fermentation, there are also a large number of bacterial fragments, which takes too long. In addition, due to the complex and inefficient process of obtaining this commercial enzyme, the commercial enzyme is expensive, with a price of €130 for 10U. Currently, there are no other heterologously expressed DSN commercial enzymes on the market. In addition, since the structural principle mechanism of the natural high temperature tolerance of the DSN enzyme from Kamchatka crabs has not yet been resolved, the heterologous cloning and transformation of the DSN enzyme have been slow, and the inability to efficiently produce heat-resistant DSN products is also one of the limiting factors.

[0005] In addition, the DSN enzyme from Antarctic krill that has obtained patent rights in the prior art has poor thermal stability, and the optimal reaction temperature is 35-40°C. At the same time, there is no commercial product of this enzyme on the market. In addition, the DSN enzyme from Scylla pseudoburrows expressed in Escherichia coli has extremely low enzyme activity at 55-60°C, and is expressed in inclusion bodies, with a complex purification process, which is not suitable for industrial production. In addition to the enzymes mentioned above, there is currently no heterologous cloned DSN enzyme on the market. Therefore, how to obtain a DSN that is easy to industrialize and has high thermal stability is very important for the market development of this enzyme. Summary of the invention

[0006] The main purpose of the present invention is to provide a preparation method of a double-stranded specific nuclease and its application, so as to solve the problem that the prior art cannot produce DSN with good thermal stability in a large amount and in a simple and convenient manner.

[0007] In order to achieve the above-mentioned object, according to the first aspect of the present invention, a method for degrading DNA in a double-stranded nucleic acid is provided, the method comprising using a double-stranded-specific nuclease to degrade the DNA in the double-stranded nucleic acid, the double-stranded-specific nuclease having an amino acid sequence as shown in SEQ ID NO:1.

[0008] Further, the double-stranded nucleic acid comprises at least one DNA chain; preferably, the double-stranded nucleic acid comprises: linear double-stranded DNA, circular double-stranded DNA and DNA-RNA hybrid; preferably, the reaction temperature of the degradation reaction is 35-55°C; preferably, the reaction temperature of the degradation reaction is 40-55°C; preferably, the reaction temperature of the degradation reaction is 50-55°C; preferably, the reaction time of the degradation reaction is 10-30min; preferably, in the degradation reaction, the final concentration of the double-stranded specific nuclease in the reaction system is 0.1-1U / μL; preferably, in the degradation reaction, the final concentration of the double-stranded nucleic acid in the reaction system is 0.1-0.5mM; preferably, the reaction system of the degradation reaction comprises: Tris-HCl with a final concentration of 50-80mM and MgCl2 with a final concentration of 5-8mM.

[0009] In order to achieve the above-mentioned purpose, according to the second aspect of the present invention, a method for detecting SNP is provided, the method comprising: a) mixing a sample to be tested, a double-stranded specific nuclease and a fluorescent probe; b) performing a degradation reaction; c) detecting the fluorescent signal and analyzing the SNP information of the sample to be tested; wherein the double-stranded specific nuclease has an amino acid sequence as shown in SEQ ID NO:1.

[0010] Furthermore, the reaction temperature of the degradation reaction is 35-55°C; preferably, the reaction temperature of the degradation reaction is 45-55°C; preferably, the reaction temperature of the degradation reaction is 50-55°C; preferably, the reaction time of the degradation reaction is 10-30min; preferably, the concentration of double-stranded specific nuclease added to the reaction system in the degradation reaction is 0.1-1U / μL; preferably, the concentration of the sample to be tested added to the reaction system in the degradation reaction is 100-200nM; preferably, the concentration of the signal probe added to the reaction system in the degradation reaction is 10-15μM; preferably, the fluorescent probe includes wild-type and mutant fluorescent probes containing different fluorescent groups; preferably, the fluorescent probe is a fluorescent probe containing a 5'-end fluorescent group and a 3'-end quenching group.

[0011] To achieve the above object, according to the third aspect of the present invention, there is provided a use of a double-stranded specific nuclease having an amino acid sequence as shown in SEQ ID NO: 1 in degrading DNA in a double-stranded nucleic acid, wherein the double-stranded nucleic acid contains at least one DNA chain.

[0012] Furthermore, the application includes RNA sequencing: mixing the sample to be sequenced with a double-stranded specific nuclease to obtain a purified RNA sample; and performing RNA sequencing on the purified RNA sample.

[0013] Furthermore, the application includes one of genomic DNA normalization, cDNA deletion and ribosomal cDNA deletion, cDNA subtraction, SNP detection, repeat-free FISH probe, miRNA multiplex fluorescence detection or telomere end quantitative detection.

[0014] In order to achieve the above object, according to a fourth aspect of the present invention, there is provided a use of a protein having an amino acid sequence shown in SEQ ID NO: 1 as a double-stranded specific nuclease.

[0015] In order to achieve the above object, according to the fifth aspect of the present invention, an isolated nucleic acid is provided, which encodes the protein having the above amino acid sequence in the above application.

[0016] In order to achieve the above object, according to the sixth aspect of the present invention, a recombinant plasmid is provided, wherein the recombinant plasmid is connected to the above isolated nucleic acid.

[0017] In order to achieve the above object, according to the seventh aspect of the present invention, a host cell is provided, in which the above recombinant plasmid is transformed.

[0018] In order to achieve the above-mentioned purpose, according to the eighth aspect of the present invention, a method for preparing a double-stranded specific nuclease is provided, which comprises: a) cloning a gene encoding a double-stranded specific nuclease having an amino acid sequence shown in SEQ ID NO:1 into an expression vector to obtain a recombinant vector; b) introducing the recombinant vector into a host cell to obtain a recombinant strain; c) culturing and inducing the recombinant strain to express the double-stranded specific nuclease to obtain a culture fluid; d) separating and extracting from the culture fluid to obtain the double-stranded specific nuclease.

[0019] By applying the technical solution of the present invention and utilizing the double-stranded specific nuclease of the present application, DNA in double-stranded nucleic acid can be degraded, and the enzyme has high thermal stability, can be well suited for industrial production, and meets market demand. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The drawings constituting a part of the present application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0021] Figure 1 A schematic diagram of the purified DSN sample in Example 1 of the present application is shown;

[0022] Figure 2 The electrophoresis results of plasmid DNA cut by DSN sample in Example 1 of the present application are shown;

[0023] Figure 3 The electrophoresis results of DSN sample cutting single-stranded DNA in Example 1 of the present application are shown;

[0024] Figure 4 A schematic diagram showing the fluorescence results of cutting DNA / RNA hybrid double strands at different temperatures of the DSN sample of the swimming crab trituberculate in Example 1 of the present application is shown;

[0025] Figure 5 A schematic diagram showing the fluorescence results of cutting DNA / RNA hybrid double strands of the DSN sample of the offshore swimming crab at different temperatures in Example 1 of the present application is shown;

[0026] Figure 6 A schematic diagram showing the fluorescence results of the DSN standard under different enzyme amounts in Example 1 of the present application is shown;

[0027] Figure 7 The standard curve of the amount of DSN standard enzyme and the average cleavage rate in Example 1 of the present application is shown;

[0028] Figure 8 The enzyme amount determination curve of the DSN sample of the swimming crab trituberculate in Example 1 of the present application is shown;

[0029] Fig. 9 The results of heterozygous typing detected by the MGB double probe method in Example 2 of the present application are shown;

[0030] Fig.10 The results of heterozygous typing detected by DSN enzyme digestion method in Example 2 of the present application are shown;

[0031] Fig.11 The wild-type typing results detected by the MGB double probe method in Example 2 of the present application are shown;

[0032] Fig.12 The wild-type typing results detected by DSN enzyme digestion method in Example 2 of the present application are shown;

[0033] Fig.13 The results of detecting mutant typing by the MGB double probe method in Example 2 of the present application are shown;

[0034] Fig.14 The results of mutant typing detected by DSN enzyme digestion method in Example 2 of the present application are shown. DETAILED DESCRIPTION

[0035] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0036] As mentioned in the background technology, although the existing commercial double-stranded specific nuclease has a certain degree of thermal stability, the acquisition process is complicated and inefficient, and the cost and selling price remain high. Other non-commercial double-stranded specific nucleases not only have complex purification processes and are not suitable for industrial production, but also have low thermal stability (35-40°C), and the actual application effect is poor. Therefore, this application intends to protect the application of a DSN enzyme with good thermal stability and suitable for industrial production.

[0037] In a first typical embodiment of the present invention, a method for degrading DNA in a double-stranded nucleic acid is provided, the method comprising using a double-stranded specific nuclease to degrade the DNA in the double-stranded nucleic acid, the double-stranded specific nuclease having an amino acid sequence as shown in SE Q ID NO:1.

[0038] SEQ ID NO:1

[0039] MEFERGIFTRFVLIVLFSACGYCSDCMWDKDKDFPRYPPLILDSSFQFVLPVEENGNRVVRLALGEIVTLACPGDELENLHLVEAKARCLEDGLLMIEDSV WDMASLGCDDVKEMILRDQGACGAGDIGVLHEIGFEVLSAERFKKVLRVCFDPTSETTLYTEHLIHGASIAAKDIDSSRPSFKTSTGFFSVSMSSVYSQN SQLELMIKLLGDEDLAHQIIDTHKQLYFAKGHMSPDADFVIMANQDATYYYINAVPQWQAFNNGNWKYLEFATRDLAEKKRRDLRVYSGGWGVLELDDINGNPVEVFLGLAQEKKVVPAPAVTWKVVHDEVTNCAAAIVGVNNPHLTSAPSTLCEDLCSSLQWIDFDVADLEHGYTYCCSVADLRAAVPHVPDLGEVCLLTE.

[0040] In the process of degrading DNA in double-stranded nucleic acid in the present application, the double-stranded specific nuclease used for degradation is derived from the swimming crab trituberculate. This DSN enzyme has good thermal stability and can efficiently play its role in the process of degrading DNA at a higher temperature. In addition, this enzyme can be secreted and expressed by yeast, which is easier to carry out industrial production.

[0041] Any double-stranded nucleic acid containing DNA is applicable to the present application. In a preferred embodiment, the double-stranded nucleic acid contains at least one DNA chain.

[0042] In a preferred embodiment, the double-stranded nucleic acid includes: linear double-stranded DNA, circular double-stranded DNA and DNA-RNA hybrid, wherein the circular double-stranded DNA includes plasmid DNA.

[0043] In order to maintain the activity of the double-stranded specific nuclease and provide a suitable environment for its reaction, in a preferred embodiment, the reaction system of the degradation reaction includes: Tris-HCl with a final concentration of 50-80 mM and MgCl2 with a final concentration of 5-8 mM.

[0044] In a preferred embodiment, the final concentration of Tris-HCl in the reaction system of the degradation reaction can be 50 mM, 60 mM, 70 mM or 80 mM.

[0045] In a preferred embodiment, the final concentration of MgCl2 in the reaction system of the degradation reaction can be 5mM, 6mM, 7mM or 8mM.

[0046] The double-stranded specific nuclease of the present application has high thermal stability. In order to further provide suitable conditions for the degradation reaction, in a preferred embodiment, the reaction temperature of the degradation reaction is 35-55°C; preferably, the reaction time of the degradation reaction is 10-30min.

[0047] In a preferred embodiment, the reaction temperature of the degradation reaction may be 35°C, 40°C, 45°C, 50°C or 55°C.

[0048] In a preferred embodiment, the reaction time of the degradation reaction is 10 min, 15 min, 20 min, 25 min or 30 min.

[0049] To further improve the efficiency of the degradation reaction, in a preferred embodiment, in the degradation reaction, the final concentration of the double-stranded specific nuclease in the reaction system is 0.1-1U / μL; preferably, in the degradation reaction, the final concentration of the double-stranded nucleic acid in the reaction system is 0.1-0.5μM.

[0050] In a preferred embodiment, the final concentration of the double-stranded specific nuclease in the reaction system in the degradation reaction can be 0.1U / μL, 0.2U / μL, 0.3U / μL, 0.4U / μL, 0.5U / μL, 0.6U / μL, 0.7U / μL, 0.8U / μL, 0.9U / μL or 1U / μL.

[0051] In a preferred embodiment, the final concentration of the double-stranded nucleic acid in the reaction system during the degradation reaction can be 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM or 0.5 μM.

[0052] In a second typical embodiment of the present invention, a method for detecting SNP is provided, the method comprising: a) mixing a sample to be tested, a double-stranded specific nuclease and a fluorescent probe; b) performing a degradation reaction; c) detecting the fluorescent signal and analyzing the SNP information of the sample to be tested; wherein the double-stranded specific nuclease has an amino acid sequence as shown in SEQ ID NO:1.

[0053] Commonly used SNP detection methods in the prior art include sequencing and Taqman-MGB probe method. Among them, the Taqman-MG B probe method is to specifically anneal and bind two fluorescent probes that detect different bases to complementary sequences during the PCR process. When the probe exists in a complete form, the fluorescent group only emits weak fluorescence due to energy resonance transfer. After the fluorescent probe hybridizes with the corresponding allele, the DNA polymerase exerts 5' exonuclease activity, and while cleaving the nucleotides one by one, it also cuts off the reporter fluorescent group, thereby emitting fluorescence. The binding of mismatched fluorescent probes will not cause cutting, and there will be no fluorescent signal. The fluorescent probe carries an MGB molecule that can bind to the minor groove of the DNA helix, further stabilizing the hybridization stability of the fluorescent probe and the template, so that fluorescent probes as short as 13 bases can obtain high mismatch discrimination capabilities.

[0054] The use of the double-stranded specific nuclease of the present application for SNP detection does not require the use of DNA polymerase for detection during the PCR reaction. It is only necessary to incubate the amplified single-stranded DNA containing the SNP site with the fluorescent probe and the double-stranded specific nuclease of the present application, and perform fluorescence detection to obtain the SNP information. This detection method can be applied to detect unpurified PCR products and two alleles in the same test tube. And the specific cellular location of this SNP information can be observed by visualizing the specific fluorescent signal. Furthermore, since the detection operation of the present application is relatively simple, it is easier to apply to SNP analysis microchips, and SNP detection can be performed more efficiently and accurately with high throughput.

[0055] The double-stranded specific nuclease of the present application has high thermal stability. In order to further provide suitable conditions for the degradation reaction, in a preferred embodiment, the reaction temperature of the degradation reaction is 35-55°C; preferably, the reaction time of the degradation reaction is 10-30min.

[0056] In a preferred embodiment, the reaction temperature of the degradation reaction may be 35°C, 40°C, 45°C, 50°C or 55°C.

[0057] In a preferred embodiment, the reaction time of the degradation reaction is 10 min, 15 min, 20 min, 25 min or 30 min.

[0058] To further improve the efficiency of the degradation reaction, in a preferred embodiment, the concentration of the double-stranded specific nuclease added to the reaction system in the degradation reaction is 0.1-1U / μL; preferably, the concentration of the sample to be tested added to the reaction system in the degradation reaction is 100-200nM; preferably, the concentration of the signal probe added to the reaction system in the degradation reaction is 10-15μM.

[0059] In a preferred embodiment, the concentration of the double-stranded specific nuclease added to the reaction system in the degradation reaction can be 0.1U / μL, 0.2U / μL, 0.3U / μL, 0.4U / μL, 0.5U / μL, 0.6U / μL, 0.7U / μL, 0.8U / μL, 0.9U / μL or 1U / μL.

[0060] In a preferred embodiment, the concentration of the sample to be tested in the degradation reaction in the reaction system can be 100nM, 110nM, 120nM, 130nM, 140nM, 150nM, 160nM, 170nM, 180nM, 190nM or 200nM.

[0061] In a preferred embodiment, the concentration of the signal probe added to the reaction system in the degradation reaction can be 10 μM, 11 μM, 12 μM, 13 μM, 14 μM or 15 μM.

[0062] The probe for fluorescence detection in the present application can simultaneously detect mutant and wild-type genotyping. In a preferred embodiment, the fluorescent probe includes wild-type and mutant fluorescent probes containing different fluorescent groups. In order to control the fluorescence generation timing of the fluorescent probe, in a preferred embodiment, the fluorescent probe is a fluorescent probe containing a 5' end fluorescent group and a 3' end quenching group. Among them, the quenching group is close to the fluorescent group and can prevent the fluorescent group from emitting light. After the fluorescent group is cut and released, it is away from the quenching group and emits fluorescence, which is captured by the fluorescence detection device.

[0063] In a third typical embodiment of the present invention, there is provided an application of a double-stranded specific nuclease having an amino acid sequence as shown in SEQ ID NO: 1 in degrading DNA in a double-stranded nucleic acid, wherein the double-stranded nucleic acid contains at least one DNA chain. The application includes detecting or purifying RNA, specifically digesting DNA in double-stranded nucleic acids in the environment to remove DNA impurities in the environment; and due to its characteristic of not cutting RNA, it can further achieve the technical purpose of purifying RNA. Therefore, any related application that requires the removal of DNA impurities in double-stranded nucleic acids, or purification to obtain a relatively pure RNA product, is applicable to the present application.

[0064] In a preferred embodiment, the application includes RNA sequencing: mixing the sample to be sequenced with a double-stranded specific nuclease to obtain a purified RNA sample; performing RNA sequencing on the purified RNA sample. In order to obtain a relatively pure sample that can be sequenced with RNA, the double-stranded specific nuclease of the present application can be used to remove DNA impurities from the sample to be sequenced before reverse transcription to obtain a higher purity RNA sample for subsequent RNA sequencing. Subsequent RNA sequencing includes sequencing the cDNA library after reverse transcription of RNA, and directly using RNA for nanopore sequencing.

[0065] In a preferred embodiment, the application also includes genomic DNA normalization, cDNA deletion and ribosomal cDNA deletion, cDNA subtraction, SNP detection, repeat-free FISH probes, miRNA multiplex fluorescence detection or telomere end quantitative detection.

[0066] In a fourth typical embodiment of the present invention, a protein having an amino acid sequence as shown in SEQ ID NO: 1 is provided for use as a double-stranded specific nuclease.

[0067] In a fifth typical embodiment of the present invention, an isolated nucleic acid is provided, which encodes a protein having the above amino acid sequence in the above application. Each host cell for protein expression has a certain preference for codons during the translation process. In order to obtain a protein product with a higher expression level in a specific host cell, the nucleotide sequence encoding the protein can be codon optimized.

[0068] In a sixth typical embodiment of the present invention, a recombinant plasmid is provided, wherein the recombinant plasmid is connected to the above-mentioned isolated nucleic acid.

[0069] In a seventh typical embodiment of the present invention, a host cell is provided, into which the above-mentioned recombinant plasmid is transformed.

[0070] In an eighth typical embodiment of the present invention, a method for preparing a double-stranded specific nuclease is provided, the preparation method comprising: cloning a gene encoding a double-stranded specific nuclease of an amino acid sequence shown in SEQ ID NO: 1 into an expression vector to obtain a recombinant vector; introducing the recombinant vector into a host cell to obtain a recombinant strain; culturing and inducing the recombinant strain to express the double-stranded specific nuclease to obtain a culture solution; separating and extracting from the culture solution to obtain the double-stranded specific nuclease. By expressing the codon-optimized nucleotide sequence of the present application in a suitable host cell, the double-stranded specific nuclease of the present application can be purified more conveniently, which provides the possibility for subsequent industrial production.

[0071] Any vector and host cell capable of expressing double-stranded specific nuclease are suitable for the present application. In a preferred embodiment, the expression vector includes: pPIC9K, pPICZαA, pHIL-S1 or pYAM75P6; preferably, the host cell is selected from any one of the following: Pichia pastoris, Hansenula polymorpha, Saccharomyces cerevisiae or Escherichia coli.

[0072] The present application is further described in detail below in conjunction with specific embodiments. These embodiments should not be construed as limiting the scope of protection claimed in the present application.

[0073] Example 1

[0074] 1. Molecular cloning

[0075] 1.1 Gene synthesis and vector construction

[0076] The gene sequence of the swimming crab was obtained from NCBI, and after codon optimization based on the Pichia pastoris host, it was sent to Universal Bio for gene synthesis to obtain the DSN amplification template. The DSN sequence was amplified using Q5 high-fidelity polymerase (NEB), and the 6HIS tag for purification was added to the C-terminus of the DSN sequence using primers. The amplified fragment and the pPIC9K vector were double-digested with EcoR I and Not I. The target gene and the linear vector fragment were connected using T4 DNA ligase and transformed into DH5α competent cells. Appropriate transformants were selected for identification, positive clones were activated, plasmids were extracted, and sent for sequencing to obtain the correct pPIC9K-DSN.

[0077] 2. Expression and Purification of DSN

[0078] 2.1 Construction and expression of recombinant expression bacteria

[0079] The pPIC9K-DSN plasmid was linearized with SalI single enzyme digestion and electroporated into Pichia pastoris GS115 competent cells. Appropriate transformants were selected for colony PCR identification. The positive transformants were GS115-pPIC9K-DSN recombinant strains. The positive monoclonal strains were activated in YPD medium, cultured overnight at 30°C, 300rpm, and the seed solution was transferred to BMMY medium. The initial OD600 was 1, and 1% methanol was added for induction. The culture was cultured at 30°C, 300rpm, and 1% methanol was added every 24 hours for a total of 96 hours.

[0080] 2.2 Fermentation supernatant collection and DSN purification

[0081] The fermentation broth was collected and centrifuged, and the supernatant was collected and purified by nickel column and anion chromatography column. After dialysis, it was stored in a storage buffer of 50mM Tris, 50% glycerol (v / v), pH 8.0. The purification results are shown in Figure 1shown.

[0082] 3. DSN activity detection

[0083] 3.1 Identification of active functions

[0084] 200 ng of plasmid DNA sample (dsDNA) and DSN were added to the reaction buffer to prepare a 10 μL reaction system, mixed well, incubated at 55°C for 10 minutes, and identified by electrophoresis. Figure 2 Lane M is a 10 kb DNA size marker; Lane 1 is the control sample DNA (without DSN); Lane 2 is the DNA treated with DSN.

[0085] 200 ng of single-stranded DNA sample (ssDNA) and DSN were added to the reaction buffer to prepare a 10 μL reaction system, mixed well, incubated at 55°C for 10 minutes, and then subjected to electrophoresis identification. Figure 3 Lane M is a 2 kb DNA size marker; Lane 3 is the single-stranded DNA of the control sample (without DSN); Lane 4 is the single-stranded DNA treated with DSN.

[0086] From the results, it can be seen that the recombinant DSN of Swimming crabs trituberculate can completely digest double-stranded DNA at 55℃, but has no obvious hydrolysis activity on single-stranded DNA, indicating that the recombinant DSN of Swimming crabs trituberculate expressed by Pichia pastoris meets the function of DSN, and the recombinant protein is expressed and purified successfully.

[0087] Among them, the 10μL reaction system for active function identification is shown in the following table:

[0088]

[0089] 3.1.1 Enzyme activity calibration by national standard method

[0090] 3.1.1.1. Experimental steps: Document reference: "Deoxyribonuclease activity detection method" (GB / T 34801-2017)

[0091] 3.1.1.2. Definition of deoxyribonuclease activity unit: the amount of enzyme required to increase the absorbance of the reaction solution at 260nm by 0.001 within 1 minute at 35℃ and pH 5.0, using 1.5mL calf thymus DNA with a concentration of 0.15mg / mL as substrate.

[0092] 3.1.1.3. Test data:

[0093]

[0094]

[0095] According to the formula The enzyme activity can be calculated to be 914U / mL.

[0096] 3.1.2 Probe method for enzyme activity calibration

[0097] 3.1.2.1. Detection principle

[0098] Based on the property that DSN can cut DNA in DNA / RNA hybrid double strands but not RNA, a DNA fluorescent probe (Taqman probe) containing a 5' end fluorescent group and a 3' end quenching group that is completely complementary to the RNA sequence is designed. The two form a hybrid double-stranded structure under appropriate conditions, and DSN specifically cuts the DNA fluorescent probe to release the fluorescent group, while RNA is not recognized and cut by DSN and can combine with the uncut DNA fluorescent probe in the system to form a hybrid double strand and enter the next round of cutting reaction.

[0099] Theoretically, the activity of DSN is positively correlated with the fluorescence signal generated by cutting the fluorescent probe, so by comparing the intensity of the generated fluorescence signal, the activity of DSN can be detected. Therefore, in the appropriate enzyme amount range and when the Taqman probe is in excess of RNA, the average DSN cutting rate is positively correlated with the enzyme activity within a certain time range.

[0100] 3.1.2.2. Determination of optimal reaction temperature

[0101] The target RNA sample sequence is SEQ ID NO: 2: UAACACUGUCUGGUAAAGAUGG, the Taqman pr obe primer P is SEQ ID NO: 3: FAM-CCATCTTTACCAGACAGTGTTA-BHQⅠ, and the DSN sample to be tested is prepared according to the following table:

[0102] system Dosage Final concentration 500 mM Tris-HCl, pH 8.0 3 μL 50mM 10mM DTT 3 μL 1mM <![CDATA[50mM MgCl2]]> 3 μL 5mM RNase inhibitor (45U / μL) 0.4μL 0.6 U / μL Taqman probe P (1μM) 3μL 100nM DSN enzyme samples 1μL 0.1U / μL Target (1μM) 3μL 100nM Sterile water Make up to 30 μL / Total volume 30μL /

[0103] Thermo Fisher ABI 7500 fast real-time fluorescence quantitative PCR instrument was used to set the reaction conditions at 35 / 40 / 45 / 50 / 55 / 60℃, 1 min per cycle, and 60 cycles in total to measure the samples. The negative control group was a sample component without DSN, and the blank control was a sample component without target. The fluorescence results of cutting DNA / RNA hybrid double strands at different temperatures are shown in Figure 2. Figure 4 shown.

[0104] from Figure 4It can be seen that when the enzyme amount is 0.1U and the Taqman probe is in excess of RNA, the activity of DSN is positively correlated with the fluorescent signal generated by the cutting of the fluorescent probe, and the average cutting rate of DSN is positively correlated with the enzyme activity. Therefore, it can be obtained from the data that the optimal reaction temperature of the recombinant swimming crab DSN expressed by yeast clone is 50℃, and the activity is weak at 60℃.

[0105] In addition, the optimal reaction temperature was detected using the DSN of the swimming crab (GenBank: ADZ58187.1) as a comparative example. Figure 5 It can be seen that the optimal reaction temperature of the recombinant swimming crab DSN expressed by yeast clone is 40℃, and it is almost inactivated at 60℃. Its thermal stability is poorer than that of the swimming crab DSN.

[0106] 3.1.2.3. Standard curve drawing

[0107] Set up a standard (Duplex-specific, evrogen, EA003) enzyme amount gradient, and dilute the samples to different multiples to establish a standard curve of DSN standard enzyme amount and average cutting rate. Set up samples at different dilution multiples, and react under the same reaction system conditions as 3.1.2.2 (reaction temperature is 50°C, and the amount of DSN enzyme samples varies according to the experimental group) to obtain the average rate. The enzyme amount of each sample (such as Figure 6-7 shown).

[0108] Among them, the enzyme amount range and time range for the samples with different dilution multiples and the reaction under the same conditions are set as follows:

[0109] ① Enzyme amount range of standard curve: Prepare DSN with different enzyme amounts (DSN enzyme sample enzyme amounts are 0, 0.001, 0.005, 0.01, 0.015, and 0.02U, respectively), and explore the maximum and minimum enzyme amounts that can be detected within the linear range of the standard curve;

[0110] ② Enzyme detection time range: Set the gradient reaction time (such as 30min, 60min, 90min, 120min), carry out enzyme-catalyzed reaction under DSN conditions with different enzyme amounts, prepare standard curves related to different DSN enzyme amounts and reaction rates in different time intervals, and confirm in which time interval the prepared standard curve has higher credibility. Figure 6 This is a schematic diagram of the fluorescence results of the DSN standard under different enzyme amounts when the reaction time is 120 min, and 4 sets of repeated experiments were performed for each enzyme amount.

[0111] 3.1.2.4. Determination of sample enzyme content

[0112] Set up the enzyme amount gradient of DSN samples of Swimming crab trituberculatus, dilute the samples into different multiples, design the reaction temperature to be the optimal temperature of 50°C, and use different concentrations of DSN to target DNA / RNA substrates for enzyme catalysis. Calculate the average reaction rate of different samples within the linear reaction range of DSN samples and substitute it into the standard curve (such as Figure 7 As shown), the amount of enzyme is calculated. Figure 8 It can be seen that the reaction rate of the sample DSN diluted 120 times is equivalent to that of the 0.02U standard, so it is calibrated to 2.4U / μL.

[0113] Example 2 Application of DSN

[0114] 1.1.SNP detection

[0115] 1.1.1. Principle of SNP detection

[0116] Single nucleotide polymorphisms (SNPs) are caused by point mutations in the base of a single nucleotide at a specific nucleotide position in genomic DNA, which causes differences between groups and individuals and forms genetic markers. SNP analysis is mainly used in genetic disease diagnosis and analysis, drug design, individual identification and paternity testing. The commonly used SNP typing test, such as the Taqman-MGB probe method, is that during the PCR process, two probes can specifically anneal and bind to the complementary sequence between the forward primer and the reverse primer. When the probe exists in a complete form, the fluorescent group only emits weak fluorescence due to energy resonance transfer. After the specific probe hybridizes with the corresponding allele, the DNA polymerase exerts 5' exonuclease activity to cut off the reporter fluorescent group, thereby emitting fluorescence. The mismatched form of the SNP probe will not cause cutting and there is no fluorescent signal. The MGB molecule binds to the minor groove of the DNA helix, and improves the hybridization stability by stabilizing the MGB probe / template, so that the probe as short as 13 bases can obtain high mismatch discrimination ability. The MGB probe includes a non-fluorescent quenching group (NFQ).

[0117] A new SNP detection method based on DSN enzyme (duplex-specific nuclease preference, DSNP) is to mix the PCR product of the DNA region containing the SNP site with the signal probe (mutant & wild type), and then add DSN for incubation. Only the duplex that completely matches between the DNA template and the signal probe will be cut by DSN to produce sequence-specific fluorescent signals, thereby achieving the purpose of detection and analysis. This method has the advantages of using unpurified PCR products as samples, analyzing two alleles simultaneously in one test tube, visualizing specific fluorescent signals, and being able to be used for the development of SNP analysis microchips;

[0118] Experimental procedures

[0119] 2.1.1.1. SNP site sample preparation

[0120] Methylenetetrahydrofolate reductase (MTHFR) is one of the key enzymes in the folate metabolism system. rs1801133 and rs1801131 are two important polymorphic sites of the MTHFR gene. The rs1801133 polymorphism causes the 222nd amino acid of the protein encoded by the MTHFR gene to change from Ala to Val (this site is also called MTHF R C677T), and the rs1801131 polymorphism causes the 429th amino acid of the protein encoded by the MTHFR gene to change from Glu to Al a (this site is also called MTHFR A1298C). These two polymorphisms will lead to a decrease in MTHFR enzyme activity and thermal stability, reduce the bioavailability of folic acid in the body, and increase the plasma homocysteine ​​(Hcy) concentration, thereby inducing a variety of diseases such as birth defects, congenital abortion, cardiovascular and cerebrovascular diseases, etc.

[0121] The MTHFR C677T site was selected for SNP analysis, and the upstream and downstream primers F01 of the amplicon (length 92 bp) region were designed to be SEQ ID NO: 4: CACAAAGCGGAAGAATGTGTCAG, and R01 was SEQ ID NO: 5: TGACCTGAAGCACTTGAAGGAGAA. The human genome sample was used as a template to amplify the region and obtain a sample.

[0122] 2.1.1.2. Design two probes with different sequences (mutant & wild type)

[0123] MGB probe: wild type F02 is SEQ ID NO: 6: CGGGAGCCGATTT-FAM, mutant 034-P-M3 is SEQ ID NO: 7: ATGAAATCGACTCCC-VIC.

[0124] 2.1.1.3.SNP detection system preparation

[0125] Preparation of SNP detection system (30 μL), prepare the reaction system according to the following table

[0126] Components concentration volume DSN reaction buffer 10X 3μL DSN 0.1U / μL 0.5μL SNP probe 1 (F02) 10μM 1.6μL SNP probe 2 (034-P-M3) 10μM 1.6μL PCR template to be tested 100nM 2μL <![CDATA[H2O]]> - 21.3μL

[0127] 2.1.1.4. Detection of fluorescence signal of the reaction system at 50°C

[0128] The MGB double probe method and DSN restriction method were used to genotype the SNP loci related to folate metabolism. Figure 9-10 As shown; wild type as Figure 11-12 As shown; mutants as Figure 13-14 As shown. Figure 9-14 It can be seen that the typing results of the DSN enzyme cutting method of the present application are consistent with those of the MGB double probe method, and the differentiation effect is good. Among them, the MGB double probe method is relatively mature and does not set up a control group. The DSN enzyme cutting method of the present application sets up a blank control, and the reaction system in the blank control is only free of the DSN enzyme component compared with the experimental group.

[0129] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: the DSN of the swimming crab obtained by the present invention is recombinantly secreted and expressed by Pichia pastoris, the purification process is simple, and it meets the functional activity of DSN. It can hydrolyze double-stranded DNA at a temperature of 50-60°C, and has no degradation effect on single-stranded DNA and RNA. The feasibility of using the purified sample for SNP detection is verified, and it has extremely high application prospects and development value.

[0130] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for degrading DNA in a double-stranded nucleic acid, characterized in that: The method comprises using a double-stranded specific nuclease to degrade the DNA in the double-stranded nucleic acid. The double-stranded specific nuclease has an amino acid sequence as shown in SEQ ID NO:

1.

2. The method according to claim 1, characterized in that The double-stranded nucleic acid comprises at least one DNA strand; Preferably, the double-stranded nucleic acid includes: linear double-stranded DNA, circular double-stranded DNA and DNA-RNA hybrid; Preferably, the reaction temperature of the degradation reaction is 35-55°C; Preferably, the reaction temperature of the degradation reaction is 45-55°C; Preferably, the reaction temperature of the degradation reaction is 50-55°C; Preferably, the reaction time of the degradation reaction is 10-30 min; Preferably, in the degradation reaction, the final concentration of the double-stranded specific nuclease in the reaction system is 0.1-1 U / μL; Preferably, in the degradation reaction, the final concentration of the double-stranded nucleic acid in the reaction system is 0.1-0.5 μM; Preferably, the reaction system of the degradation reaction comprises: Tris-HCl with a final concentration of 50-80 mM and MgCl2 with a final concentration of 5-8 mM.

3. A method for detecting SNP, characterized in that: The method comprises: a) mixing the sample to be tested, the double-stranded specific nuclease and the fluorescent probe; b) carrying out degradation reaction; c) detecting the fluorescent signal and analyzing the SNP information of the sample to be tested; Wherein, the double-stranded specific nuclease has an amino acid sequence as shown in SEQ ID NO:

1.

4. The method according to claim 3, characterized in that The reaction temperature of the degradation reaction is 35-55°C; Preferably, the reaction temperature of the degradation reaction is 45-55°C; Preferably, the reaction temperature of the degradation reaction is 50-55°C; Preferably, the reaction time of the degradation reaction is 10-30 min; Preferably, the double-stranded specific nuclease is added to the reaction system at a concentration of 0.1-1 U / μL in the degradation reaction; Preferably, the concentration of the sample to be tested in the degradation reaction is 100-200 nM in the reaction system; Preferably, the concentration of the signal probe added to the reaction system in the degradation reaction is 10-15 μM; Preferably, the fluorescent probes include wild-type and mutant fluorescent probes containing different fluorescent groups; Preferably, the fluorescent probe is a fluorescent probe comprising a fluorescent group at the 5' end and a quenching group at the 3' end.

5. Use of a double-stranded specific nuclease having an amino acid sequence as shown in SEQ ID NO: 1 in degrading DNA in double-stranded nucleic acid, wherein: The double-stranded nucleic acid comprises at least one DNA strand.

6. The use according to claim 5, characterized in that: The applications include RNA sequencing: Mixing the sample to be sequenced with the double-stranded specific nuclease to obtain a purified RNA sample; The purified RNA samples were subjected to RNA sequencing.

7. The use according to claim 5, characterized in that: The application comprises one of genomic DNA normalization, cDNA deletion and ribosomal cDNA deletion, cDNA subtraction, SNP detection, repeat-free FISH probe, miRNA multiplex fluorescence detection or telomere end quantitative detection.

8. Use of a protein having an amino acid sequence as shown in SEQ ID NO: 1 as a double-stranded specific nuclease.

9. The use according to claim 8, characterized in that: The application also includes preparing an isolated nucleic acid, a recombinant plasmid or a host cell, wherein the isolated nucleic acid encodes a protein having an amino acid sequence shown in SEQ ID NO: 1, the recombinant plasmid is connected with the isolated nucleic acid, and the host cell is transformed with the recombinant plasmid.

10. A method for preparing a double-stranded specific nuclease, characterized in that: The preparation method comprises: a) cloning a gene encoding a double-stranded specific nuclease having an amino acid sequence as shown in SEQ ID NO: 1 into an expression vector to obtain a recombinant vector; b) introducing the recombinant vector into a host cell to obtain a recombinant strain; c) culturing and inducing the recombinant strain to express the double-stranded specific nuclease to obtain a culture solution; d) separating and extracting from the culture solution to obtain the double-stranded specific nuclease.

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