阅读说明：本技术 T7EI-like核酸酶反应缓冲体系、DNA错配检测方法及应用 (T7EI-like nuclease reaction buffer system, DNA mismatch detection method and application ) 是由 范三红 单丽伟 胡小平 于 2020-06-24 设计创作，主要内容包括：本发明公开了一种T7EI-like核酸酶反应缓冲体系、DNA错配检测方法及应用,涉及一种T7EI-like核酸酶的反应缓冲溶液配方,以及利用该缓冲体系增强该类核酸酶错配识别切割能力的应用。常规的反应缓冲体系中,T7EI-like核酸酶对插入缺失(InDel)导致的错配DNA具有较好的识别切割能力,而对单核苷酸多态性(SNP)导致的错配DNA的识别切割能力较差；本发明的反应缓冲体系中,T7EI-like核酸酶对两类错配DNA均有良好的识别切割能力。该反应缓冲体系的核心在于其特定的缓冲溶质配比、去垢剂浓度、二价金属离子及辅助蛋白配比。该发明可应用于基因编辑效率的检测、DNA样品中错误序列的消除、基因编辑个体的筛查、样本中InDel和SNP位点的检测筛选、将InDel和SNP位点转化为分子标记进行分子辅助育种。(The invention discloses a T7EI-like nuclease reaction buffer system, a DNA mismatch detection method and application, and relates to a reaction buffer solution formula of T7EI-like nuclease and application for enhancing the mismatch recognition cutting capability of the nuclease by using the buffer system. In a conventional reaction buffer system, T7EI-like nuclease has better recognition and cutting capabilities on mismatched DNA caused by insertion deletion (InDel), and has poorer recognition and cutting capabilities on mismatched DNA caused by Single Nucleotide Polymorphism (SNP); in the reaction buffer system, T7EI-like nuclease has good recognition and cutting capacity on two types of mismatched DNA. The core of the reaction buffer system is the specific mixture ratio of buffer solute, the concentration of detergent, the mixture ratio of divalent metal ions and auxiliary protein. The invention can be applied to the detection of gene editing efficiency, the elimination of error sequences in DNA samples, the screening of gene editing individuals, the detection and screening of InDel and SNP sites in samples, and the conversion of the InDel and SNP sites into molecular markers for molecular assisted breeding.)

1. A T7EI-like nuclease reaction buffer system is characterized in that the reaction buffer system comprises T7EI-like nuclease, a buffer reagent, a detergent, catalytic metal ions, a stabilizer and DNA ligase;

the buffer reagent adopts one of Tris-hydrochloric acid or Tris-acetic acid;

the detergent adopts Triton X-100 and/or Tween-20;

the metal ion of the catalytic metal ion comprises Mg²⁺、Co²⁺Or Mn²⁺One or more of;

the stabilizer comprises BSA.

2. The T7EI-like nuclease reaction buffer system of claim 1, wherein the T7EI-like nuclease comprises T7EI nuclease from e.coli T7 phage and other nucleases homologous to T7EI nuclease and having catalytic properties similar thereto; or include P-SSP7EI nuclease from cyanobacterial P-SSP7 phage, and other nucleases homologous to P-SSP7EI nuclease and having similar catalytic properties.

3. The buffer system for T7EI-like nuclease reaction according to claim 1, wherein the buffer reagent has a final concentration of 20-50 mM and a pH of 7.4-9.0;

the detergent is used for enhancing the dispersity of enzyme protein, and the volume concentration of Triton X-100 is 0.1-0.5%, or the volume concentration of Tween-20 is 0.02-0.1%;

the metal ions are used for participating in substrate combination and catalysis, and the concentration is 2-10 mM;

the stabilizer is nonspecific protein for enhancing enzyme stability, and the concentration of the stabilizer is 10-100 ng/ml;

the DNA ligase is used for counteracting the nonspecific cleavage activity of T7EI-like nuclease, comprises one of T4-like nuclease, Taq-like nuclease and Ecoli-like nuclease, and also comprises a substrate ATP or NAD⁺。

4. A method for detecting DNA mismatches,

firstly, preprocessing a DNA sample to be detected, and sequentially performing denaturation annealing on the DNA sample to be detected or the DNA sample to be detected and a known DNA sample to form mismatched DNA and DNA precipitation;

then, adding the product obtained in the last step into a T7EI-like nuclease reaction buffer system of any one of claims 1-3, carrying out electrophoretic detection after mismatch identification and cutting after a warm bath reaction for a period of time, and judging whether the DNA sample to be detected has mismatch according to the electrophoretic detection result;

if the type of mismatched DNA is InDel type, then no DNA precipitation step is required.

5. Use of the T7EI-like nuclease reaction buffer system of claim 1 for gene editing; the T7EI-like nuclease reaction buffer system can better identify both InDel and SNP type variations, and can more comprehensively evaluate the gene editing efficiency; in the process of screening gene editing individuals, the optimized reaction buffer system can be used for screening out the SNP type variation which is possibly ignored originally.

6. Use of the T7EI-like nuclease reaction buffer system of claim 1 in a sequence variation detection, screening, tracking technology based on T7EI-like nuclease cleavage; the sources of differences between sample sequences are not limited to gene editing but also include natural individual differences or variations caused by other chemical-physical methods. When the site-directed mutagenesis is carried out by PCR, a small amount of DNA pollution before the mutagenesis is eliminated by utilizing the T7EI-like nuclease reaction buffer system; when the Tilling technology is used for screening variation of an individual in a specific gene, the T7EI-like nuclease reaction buffer system is used for enhancing the screening efficiency of T7EI-like nuclease on SNP type variation; any InDel or SNP type variation can be converted into a molecular marker by utilizing the reaction buffer system of the T7EI-like nuclease, and the molecular marker is applied to large-scale molecular breeding and excellent character tracking polymerization; theoretically, the T7EI-like nuclease reaction buffer system can be applied to research and application of discovery, screening and tracking of InDel and SNP type variation between any homologous sequences.

Technical Field

The invention belongs to the technical field of biology, relates to molecular biology, genetic engineering and biotechnology application, and particularly relates to a method for enhancing mismatch cutting capability of T7EI-like nuclease and application thereof.

Background

The difference between species and individuals is determined by the difference between the genome and the gene sequence, the fact that whether the difference exists between homologous sequences or not and the difference is judged by rapid screening is an important issue in the fields of molecular biology research and biotechnology, and related research methods and technologies not only have wide application prospects in the fields of medicine and agriculture, but also have huge market values.

Whether the homologous sequence variation is generated by natural evolution, artificial mutagenesis or directed editing, the difference between the sequences can be divided into two major categories of insertion deletion (InDel) and Single Nucleotide Polymorphism (SNP). How to detect differences between homologous sequences between different individuals? There are three methods currently in use: one method is a PCR + electrophoresis method, firstly, a target DNA fragment is obtained through PCR amplification, then whether the difference exists between the sequence length and the sequence itself or not is detected through high-resolution electrophoresis, the InDel type difference of a long fragment is relatively easy to detect, if the InDel length is too short, the InDel type difference can be distinguished through sequencing gel with extremely high resolution, the SNP type difference needs to analyze single-strand conformation polymorphism (SSCP) through denaturing electrophoresis, and the technical operation difficulty is larger. The other is PCR + mismatch cutting method, which includes PCR amplification, denaturation annealing of the DNA segment to be detected and known DNA segment, forming mismatch if there is difference between the two sequences, adding nuclease for specific recognition of mismatch site to cut, and converting mismatch signal through specific enzyme cutting. Commonly used nucleases for mismatch recognition include T7EI (T7 endonuclease I) from the T7 phage, which has a greater ability to recognize InDel-type mismatches, and CEL I nuclease from celery, which has a better ability to recognize SNPs. The third is PCR + sequencing, and the difference between the sequences can be finally determined by sequencing, but the relative cost is much higher than that of the former two. The methods have advantages and disadvantages and respective application fields.

T7EI is the encoded product of the T7 phage gene 3, a multifunctional structure-specific nuclease (NP-041972.1) that recognizes and cleaves Holiday DNA, mismatch DNA, and nick (nick) DNA. The mismatched DNA includes the aforementioned InDel type and SNP type. T7EI was most active in recognizing Holiday DNA, followed by InDel-type mismatches, which were less able to recognize SNP-type mismatches. T7EI also has random nicking (random nick) activity and nick-sitecleavage (nick-sitecleavage) activity. P-SSP7EI (P-SSP7endonuclease I, YP-214193) is a T7EI homologous protein encoded by cyanobacterial phage P-SSP7 gene 3, which has similar Holiday DNA and mismatch DNA recognition and cleavage activities as T7EI, but its nick-site cleavage activity is very different from that of T7EI [5 ].

T7EI is applied to mutation detection since the discovery, and with the increasing demand of people for sequence variation detection, gene editing technology is emerging and the application is wider. There are currently available on the market both T7EI alone and various kits developed based on this enzyme. For example, NEB, EnGen mutation detection Kit developed based on the enzyme, and Guide-it InDel Identification Kit developed by Clonetech.

T7EI is a multifunctional nuclease, and although the enzyme is widely used in mutation screening detection at present, some characteristics of the enzyme influence the application of the enzyme. Firstly, the recognition efficiency of the enzyme to different mismatched substrates is obviously different, the recognition and cutting efficiency to the Holiday DNA is the highest, secondly, the recognition effect to the SNP type mismatch is poor due to InDel type mismatch, and the enzyme has certain preference to the type of mismatched base [3 ]. In practical application, InDel and SNP type mismatching exist simultaneously, so that SNP type differences are easy to miss in detection results, and even if partial SNP type differences are detected, the results have bias. Secondly, T7EI has, in addition to specifically recognizing a specific structure in the sequence and cleaving the DNA from the site, a non-specific random nicking activity and nicking site cleavage activity, which means that DNA is slowly degraded by the activity even if there is no mismatch in the sequence, which should be inhibited or eliminated in a mismatch detection reaction.

Disclosure of Invention

The T7EI-like nuclease has low capability of identifying SNP type mismatch in mismatch cleavage reaction and has base preference; meanwhile, the enzyme has the capability of randomly degrading any DNA, and the capability can influence the mismatch recognition cutting effect. In order to solve the problems of T7EI-like nuclease in mismatched DNA detection, the invention provides a method for improving recognition and cutting of mismatched DNA by T7EI-like nuclease and a reaction buffer system, and improves and expands the application of T7EI-like nuclease in mutation detection, screening and tracking.

In order to overcome the problems in the application of T7EI-like nuclease, the invention adopts the following technical scheme:

the invention discloses a reaction buffer system of T7EI-like nuclease, which comprises the following components in percentage by weight:

a buffer system for T7EI-like (T7 endonuclease I-like) nuclease reaction comprises T7EI-like nuclease, Tris-HCl and Tris-Ac for providing buffer capacity, detergents Triton X-100 and Tween-20 for enhancing the protein dispersity of the nuclease, and metal ions Mg for binding and catalyzing substrates²⁺、Co²⁺、Mn²⁺And the like, including nonspecific proteins such as BSA which enhance enzyme stability, and DNA ligase which counteracts nonspecific cleavage activity, and the like.

The T7EI-like nuclease comprises T7EI (T7 endonuclease I), P-SSP7EI (P-SSP7endonuclease I) and homologous proteins thereof; the protein can be obtained from commercial products through expression and purification, can be an enzyme protein, and can also be a fusion protein of T7EI-like protein, such as T7EI fused with Maltose Binding Protein (MBP), and the activity of the protein needs to be calibrated before application;

the buffer capacity provider is Tris-HCl or Tris-Ac or other buffers, the final concentration of the buffer reagent is 20-50 mM, and the pH value is 7.4-9.0;

the detergent for enhancing the dispersity of the enzyme protein comprises Triton X-100 and Tween-20, wherein the concentration of the Triton X-100 is 0.1-0.5%, and the concentration of the Tween-20 is 0.02-0.1%;

the metal ions involved in substrate binding and catalysis include Mg²⁺、Co²⁺、Mn²⁺Etc. in a concentration of 2 to 10 mM;

the nonspecific protein for enhancing the enzyme stability is BSA and the like, and the concentration is 10-100 ng/ml;

the protein for counteracting the non-specific cleavage activity of the T7EI-like nuclease is mainly DNA ligase, such as T4-ligase, Taq-ligase, Ecoli-ligase and the like, and various substrates corresponding to the ligase, such as ATP or NAD, need to be added⁺。

When the enhanced T7EI-like nuclease mismatch recognition cleavage reaction buffer system is applied, a detected DNA sample needs to be pretreated firstly, and the main aim of pretreatment is to form mismatch DNA and remove various substances in the original buffer system. The pretreatment usually involves two steps, denaturation annealing and DNA precipitation, the first step is used to form mismatched DNA, and the second step is used to remove various buffer reagents, ions and proteins in the original buffer system. Then adding the buffer solution and the T7EI-like nuclease disclosed by the invention, carrying out warm bath for a proper time (such as 20-30 minutes) at the optimal reaction temperature (such as 37 ℃) of the enzyme, then carrying out separation by utilizing agarose gel electrophoresis or polyacrylamide gel electrophoresis, and judging whether the original DNA sample has mismatch according to the electrophoresis result.

The method is a universal mismatch DNA detection method, can be applied to various fields, such as detection of gene editing efficiency, screening of gene editing individuals, screening of specific known variation, discovery of whether variation exists in DNA, screening of different variations of known genes by using T7EI-like nuclease and the reaction system to replace celery CEL I nuclease in Tiling, converting any InDel and SNP variation into molecular markers by using the technology in molecular breeding, and tracking specific genotypes in offspring, thereby realizing molecular-assisted gene polymerization and molecular design.

The invention has the outstanding advantages that:

the T7EI-like nuclease reaction buffer system disclosed by the invention can obviously improve the capability of identifying and cutting SNP type mismatching DNA of T7EI-like nuclease, overcome the defect of T7EI-like nuclease in mutation detection and expand the application of T7EI-like nuclease.

The technical method disclosed by the invention can reduce the capacity of T7EI for non-specific DNA cleavage and highlight the capacity of T7EI for recognizing and cleaving mismatched DNA.

Drawings

FIG. 1 is a basic application flow of an enhanced T7EI-like nuclease mismatch recognition cleavage reaction system.

FIG. 2 shows the results of electrophoretic analysis of T7EI recognition of different mismatches in normal and enhanced reaction buffers.

Detailed Description

In order to make the embodiments of the present invention specifically disclosed, the following description is made only by way of examples and accompanying drawings, and it should be understood that the present invention is not further limited thereto.

It should be noted that: the T7EI-like nuclease comprises T7 endonuclease I (T7EI) nuclease which is relatively well researched and is derived from Escherichia coli T7 phage, and other nucleases which are homologous with the T7EI nuclease and have similar catalytic properties, for example; phiA1122 endonuclease I (phiA1122EI) nuclease from Yersinia phage phiA 1122. Including the P-SSP7Endonuclease I (P-SSP7EI) nuclease from the cyanobacterial P-SSP7 phage, as well as other nucleases homologous to the P-SSP7EI nuclease and similar in catalytic properties, such as Syn5 Endonuclease I (Syn5EI) from the cyanobacterial phage Syn 5. T7EI and P-SSP7EI are listed separately because they are homologous and have similar catalytic properties, but the sequence identity is only about 30%, and they are easily classified as two types of nucleases. The protein can be obtained from commercial products, can be obtained by expression and purification, can be an enzyme protein per se, and can also be a fusion protein of T7EI-like protein, such as T7EI fused with Maltose Binding Protein (MBP), and the activity of the protein needs to be calibrated before application.

In addition, the final concentration of the buffer reagent according to the present invention is the final concentration of each component after the final mixing.

The basic application flow of the disclosed technology is shown in fig. 1, and the specific embodiment is as follows: