A method for rapidly obtaining cleavage patterns of unknown endonucleases

Abstract

The application discloses a method for quickly obtaining a cleavage mode of unknown endonuclease, which comprises the following steps: firstly, connecting DNA fragments obtained by cleavage of unknown endonuclease into a T vector after treatment; secondly, performing sequencing on the connection sites of the connection products in the direction of the connection sites by using sequencing primers, so that the sequencing results cover the breaking positions of the substrate DNA; and thirdly, according to the sequence information of the exogenous DNA fragments (i.e. original DNA fragments) and the sequences adjacent to the T vector boundary, the breaking positions and the breaking modes of the substrate DNA fragments can be accurately inferred through sequence alignment analysis, so that the method can be used for recognition site determination of unknown endonuclease, and has the advantages of simple and quick operation, accurate result, low cost, short time consumption and the like.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically a method for rapidly obtaining the cleavage patterns of unknown nucleic acid endonucleases. Background Technology

[0002] Nucleases are widely distributed in living organisms, playing a crucial role in nucleic acid metabolism and serving as essential tool enzymes in molecular biology. Nucleases from different sources exhibit varying specificities and modes of action. Some nucleases act only on RNA and are called ribonucleases (RNases), while others act only on DNA and are called deoxyribonucleases (DNases). Still others have lower specificity, acting on both RNA and DNA, and are collectively referred to as nucleases. Based on their site of action, nucleases can be further classified into exonucleases and endonucleases. In the 1970s, a class of endonucleases was discovered in bacteria that specifically recognize and hydrolyze specific nucleotide sequences on double-stranded DNA; these are called restriction endonucleases (or simply restriction enzymes). When foreign DNA invades bacteria, restriction endonucleases hydrolyze it into fragments, thus restricting the expression of foreign DNA within the bacterial cell. The bacterial DNA itself, modified by methylases at specific nucleotide sequences, is protected from hydrolysis. Research and application of restriction endonucleases have developed rapidly; more than 100 types of restriction endonucleases have been purified, many of which have become indispensable tools in genetic engineering research and are widely used in DNA cloning and sequencing. Endonucleases act on the phosphodiester bonds of double-stranded DNA, recognizing specific base sequences and having specific cleavage sites. Determining the cleavage specificity of unknown endonucleases is crucial for their research and utilization. Therefore, accurately obtaining their cleavage patterns is essential for better studying the functional characteristics of unknown endonucleases.

[0003] In the prior art, Chinese patent CN109207571A discloses a method for detecting endonuclease cleavage sites. This method utilizes billions of nucleic acid sequences on a chip to systematically detect the recognition and cleavage sites and core sequence characteristics of endonucleases on DNA in a high-throughput manner. It employs two sequencing steps to obtain the base sequence and normal double-stranded DNA, respectively. Simultaneously, based on the changes in fluorescence signals before and after enzyme digestion, bioinformatics analysis methods are used to determine the endonuclease recognition and cleavage sites. By studying the endonuclease recognition and cleavage sites, the tendency of endonucleases to recognize and cleave these sites can be detected, and the star activity of endonucleases and off-target effects of endonucleases in genome editing technologies can be predicted in advance. While this method has the advantage of high throughput, it suffers from drawbacks such as technical difficulty, high cost, long processing time, and relatively complex operation. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a method for rapidly obtaining the cleavage pattern of unknown endonucleases. This method can accurately deduce the cleavage location and pattern of unknown endonucleases, and is used for the determination of recognition sites of unknown endonucleases. It has advantages such as simple and rapid operation, accurate results, low cost, and short processing time. Specifically, it is achieved through the following techniques.

[0005] A method for rapidly obtaining the cleavage pattern of an unknown nucleic acid endonuclease includes the following steps:

[0006] S1. The DNA substrate was specifically cleaved with an unknown nuclease to obtain several original DNA fragments, which were then recovered by gel cutting.

[0007] S2. The original DNA fragments recovered in step S1 are subjected to end-trimming and dephosphorylation.

[0008] S3. The DNA fragment obtained in step S2 is ligated into the T vector, and the DNA fragment ligated into the T vector is transformed into the host bacteria;

[0009] S4. Positive recombinants were obtained by screening, and sequencing was performed in the direction of the ligation site using sequencing primers on the T vector. Based on the boundary sequence of the T vector and the sequence of the adjacent DNA fragment, the break location and breakage mode of the original DNA fragment were inferred, that is, the cutting mode of the unknown endonuclease was obtained.

[0010] The sequencing primer is any sequence on the T vector, and the distance between the first base of the sequencing primer and the linking site is 50-800 bp, starting and ending at the base distance between the first base of the sequencing primer and the linking site.

[0011] The principle of this method is as follows: (1) The DNA substrate is cut by an unknown nuclease to obtain several original DNA fragments after cutting, and the original DNA substrate is recovered by gel cutting. The break position of the original DNA substrate is the cutting site of the unknown nuclease; (2) In order to obtain the cutting mode of the unknown nuclease, the DNA fragments obtained by gel cutting are end-aligned and dephosphorylated, and then ligated into T vectors respectively; (3) The ligation product (T vector containing the treated DNA fragments) is transformed into host bacteria (such as Escherichia coli), single clones are picked, and PCR amplification detection is performed using the amplification primers on the T vector (such as M13F and M13R) to screen out positive recombinants; (4) Sequencing primers on the T vector (such as M13F and M13R) are then used to sequence the ligation site (i.e. the ligation position of the T vector and the treated DNA fragment, i.e. the break position of the substrate DNA) respectively. Based on the information of the DNA fragment sequence and the adjacent T vector sequence, the break position and cutting mode of the unknown nuclease can be inferred through sequence alignment analysis.

[0012] Preferably, the distance between the first base of the sequencing primer and the linker site is 100-600 bp. Existing first-generation sequencing methods are inaccurate for bases with distances of 0-50 bp and greater than 800 bp, resulting in errors. This invention limits the distance to the above range, avoiding these regions and ensuring that the sequence information at the break point is within the 100-600 bp range of the sequencing result, leading to more accurate results; it also avoids errors caused by the limitations of current first-generation sequencing technology.

[0013] Preferably, the DNA substrate in step S1 is 300-2000 bp in length. The DNA fragment used as the cleavage substrate can be selected as needed, but is generally preferably around 300-2000 bp. Fragments that are too long or too short are not conducive to gel recovery and ligation efficiency. Furthermore, the selected cleavage substrate DNA fragment should ideally contain only one cleavage site, and after cleavage, two or more DNA fragments of clearly distinguishable size should be obtained to reduce workload, effectively perform gel recovery, and improve results.

[0014] Using the above-mentioned technical solution of the present invention: the sequencing primers are sequences on the T vector (such as M13F and M13R). When the length of the original DNA fragment obtained after cutting is 100-800bp, one sequencing reaction can obtain the sequences at both ends of the exogenous DNA fragment (i.e., the original DNA fragment) ligated into the T vector; when the length of the original DNA fragment obtained after cutting is >800bp, one sequencing reaction can only ensure the sequence at one end of the exogenous DNA fragment ligated into the T vector.

[0015] More preferably, the optimal length of the DNA substrate in step S1 is 600-1200 bp. In this case, the amplification primers used for screening positive clones and the sequencing primers used for detecting break positions can be set to M13F and M13R, respectively. One sequencing reaction can obtain the sequences at both ends of the exogenous DNA fragment (i.e., the original DNA fragment) ligated into the T vector, thereby simplifying the procedure, making the operation simpler, and reducing costs.

[0016] Preferably, after completing steps S1 and S2, the DNA fragments recovered in step S1 and the DNA fragments processed in step S2 are subjected to DNA purification treatment, respectively. The DNA purification treatment is performed using a DNA purification kit or the ethanol precipitation method.

[0017] Preferably, in step S2, the end-leveling treatment uses a rapid end-leveling kit. This converts the 5' or 3' protruding ends of non-level DNA into blunt ends with a 5' phosphate. The T4 DNA polymerase in the rapid end-leveling kit has both 3'→5' exonuclease and 5'→3' polymerase activities, enabling DNA end leveling. When the end is a 5' protruding end, the T4 DNA polymerase can use its 5'→3' DNA polymerase activity to level the end; when the end is a 3' protruding end, the T4 DNA polymerase can use its 3'→5' DNA exonuclease activity to trim the end. The rapid end-leveling kit is a commonly available commercially available kit in the industry, such as the Rapid End-Leveling Kit (#E1201) manufactured by NEB (New England Biolabs, Inc.).

[0018] Preferably, in step S2, the dephosphorylation treatment uses a dephosphorylase.

[0019] Preferably, the T vector in step S3 is obtained using the TOPO cloning kit.

[0020] Preferably, in step S3, the DNA fragment obtained in step S2 is ligated into the T vector using the TOPO cloning kit.

[0021] Preferably, in step S4, the specific steps for screening positive clones are as follows: select single clones from step S3, and perform PCR amplification and detection using amplification primers (e.g., M13F, M13R) on the T vector to obtain positive recombinants.

[0022] Compared with existing technologies, the advantages of this invention are: it provides a novel method for determining the cleavage site and cleavage mode of an unknown endonuclease, and no other domestic or international research institutions have been found to have adopted this method. The DNA fragment obtained from the unknown endonuclease is processed and ligated into a T-vector. Sequencing primers are used to sequence the ligation site of the ligation product, ensuring that the sequencing results cover the breakage location of the substrate DNA. Then, based on the sequence information of the exogenous DNA fragment (i.e., the original DNA fragment) and the adjacent T-vector boundary, sequence alignment analysis can accurately infer the breakage location and mode of the substrate DNA fragment. Therefore, it can be used to determine the recognition site of unknown nucleases, and has the advantages of simple and rapid operation, accurate results, low cost, and short processing time. Attached Figure Description

[0023] Figure 1 This is a flowchart illustrating the method for rapidly obtaining the cleavage pattern of an unknown nucleic acid endonuclease in Example 1. Figure I ;

[0024] Figure 2 This is a flowchart illustrating the method for detecting unknown endonuclease cleavage patterns in Example 1. Figure II ;

[0025] Figure 3 The sequencing results are for the unknown endonuclease cleavage pattern in Example 1. Detailed Implementation

[0026] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] Example 1

[0028] This embodiment employs a nuclease with an unknown cleavage mode, which can cleave a 955bp DNA fragment (DNA substrate) into two original DNA fragments of 372bp and 583bp. To obtain the cleavage mode of this nuclease, the two original DNA fragments are first gel-cleaved and recovered, then end-trimmed and dephosphorylated, and then ligated into a T-vector to obtain a ligation product. The ligation product (i.e., the T-vector containing the treated DNA fragments) is then transformed into E. coli, and single clones are selected. PCR amplification is performed using the amplification primers on the T-vector to screen for positive recombinants. Then, 10-20 positive recombinants are selected and sequenced using the sequencing primers on the T-vector to obtain the sequence information at the junction with the T-vector. This sequence is then compared with the substrate DNA sequence to accurately determine the cleavage site and cleavage mode of the nuclease. The procedure of this embodiment is attached. Figure 1 , 2 As shown, the specific steps are as follows:

[0029] S1. A DNA substrate with a length of 955 bp was specifically cleaved with an unknown nuclease to obtain two original DNA fragments with lengths of 372 bp and 583 bp, respectively, which were then recovered by gel cutting.

[0030] S11. Amplify a DNA fragment of known sequence with a length of 955 bp using PCR, and then purify the amplification product. Specifically, use the DNA purification kit (#T1030) manufactured by NEB (New England Biolabs, Inc., USA) for DNA purification.

[0031] S12. Using the DNA fragment from S11 as a reaction substrate, the fragment was cut with an unknown nuclease to obtain two specific DNA fragments of 372bp and 583bp, respectively. These fragments were then gel-cleaved, recovered, and purified.

[0032] S2. The raw DNA fragments recovered in step S1 were end-equalized using the NEB Rapid End Equalization Kit (#E1201). The reaction was carried out at room temperature for 15 min. The reaction system is shown in Table 1 below. Then, DNA purification was performed using the same method as in S11.

[0033] Table 1. Terminal smoothing and phosphorylation reaction system

[0034] DNA 19 μL (1 μg) 10×Blunting Buffer 2.5μL 1mM dNTP Mix 2.5μL Blunt Enzyme Mix 1.0μL Total volume 25μL

[0035] Dephosphorylation was then performed using NEB-produced dephosphorylase (#M0525) at 37°C for 10 min. The reaction system is shown in Table 2. DNA purification was then performed using the same method as S11.

[0036] Table 2 Dephosphorylation reaction system

[0037] DNA 1 pmol of DNA ends 10×CutSmart Buffer 2μL Quick CIP 1μL Total volume 20μL

[0038] S3. The DNA fragment obtained in step S2 was ligated into the T vector. The ligation reaction was performed using the second-generation TOPO cloning kit (#C601-01) manufactured by Novizan. The reaction system is shown in Table 3 below. The reaction was carried out at room temperature (20-37℃) for 5 min to obtain the ligation product. The ligation product (DNA fragment ligated with the T vector) was then transformed into Escherichia coli DH5α.

[0039] Table 3 Connection Reaction System

[0040] 5×Blunt Clonging Mix 1μL The DNA fragment obtained in step S2 1μL Nuclease-free Water 3μL Total volume 5μL

[0041] S4. Select the single clones from step S3, perform PCR amplification using amplification primers (M13F / M13R primer pair commonly used in this field, with known nucleotide sequences), and then perform gel electrophoresis to screen out positive recombinants. Then, use sequencing primers M13F to sequence towards the ligation site. Based on the known sequence and the adjacent plasmid sequence, determine the cutting position and cutting method of the original DNA fragment.

[0042] In this embodiment, the sequencing primer was M13F, and the sequencing results are shown in the attached figure. Figure 3 As shown. In addition, it is known to those skilled in the art that the sequencing primers are not limited to M13F and M13R, but can also be any sequence on the T vector, as long as the sequencing results obtained by using the sequencing primers can cover the junction between the T vector and the linked DNA fragment, and can ensure the accuracy of the base sequence near the junction, then the sequencing primers can achieve the technical solution of the present invention.

[0043] During the end-trimming of the original DNA fragment in step S2, different cutting methods will result in different trimming patterns. With a 5' protruding end, the DNA end will be trimmed; with a 3' protruding end, the DNA end will be trimmed; and with a blunt end, there will be no change. These three cases can be confirmed based on the sequence information at the junction of the T-vector and the exogenous DNA. Different clones are then sequenced. Figure 3 This example demonstrates the sequencing results of the unknown endonuclease producing 5' overhangs in this embodiment. Figure 3The bolded sequences marked in the figure correspond to the sequences on the exogenous DNA ligated into the T vector obtained from the sequencing reaction. Based on the above results, the cleavage pattern of the unknown endonuclease used in this embodiment can be determined as follows: positive strand 5'…AATAACC / CGGATATT…3', negative strand 5'…AATATCC / GGGTTATT…3', resulting in a double-strand break in DNA with a single nucleotide protrusion at the 5' end. When using the method of this invention to detect the cleavage pattern of the unknown endonuclease, the detection results are accurate, and the operation is simple and rapid.

[0044] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for rapidly obtaining the cleavage pattern of an unknown nucleic acid endonuclease, characterized in that, Includes the following steps: S1. DNA substrates with a length of 300-2000 bp were specifically cleaved with an unknown nuclease to obtain several original DNA fragments, which were then recovered by gel cutting. S2. The original DNA fragments recovered in step S1 are subjected to end-trimming and dephosphorylation. S3. The DNA fragment obtained in step S2 is ligated into the T vector, and the DNA fragment ligated into the T vector is transformed into the host bacteria; S4. Positive recombinants were obtained by screening, and sequencing was performed in the direction of the ligation site using sequencing primers on the T vector. Based on the boundary sequence of the T vector and the sequence of the adjacent DNA fragment, the break location and breakage mode of the original DNA fragment were inferred, that is, the cutting mode of the unknown endonuclease was obtained. The sequencing primer is any sequence on the T vector, and the distance between the first base of the sequencing primer and the linking site is 50-800 bp.

2. The method for rapidly obtaining the cleavage pattern of an unknown nucleic acid endonuclease according to claim 1, characterized in that, The distance between the first base of the sequencing primer and the linker site is 100-600 bp.

3. The method for rapidly obtaining the cleavage pattern of an unknown nucleic acid endonuclease according to claim 1, characterized in that, The optimal length of the DNA substrate described in step S1 is 600-1200 bp.

4. The method for rapidly obtaining the cleavage pattern of an unknown nucleic acid endonuclease according to claim 1, characterized in that, After completing steps S1 and S2, the DNA fragments recovered in step S1 and the DNA fragments processed in step S2 are subjected to DNA purification treatment, respectively.

5. The method for rapidly obtaining the cleavage pattern of an unknown nucleic acid endonuclease according to claim 1, characterized in that, In step S2, the end-leveling process uses a rapid end-leveling kit.

6. The method for rapidly obtaining the cleavage pattern of an unknown nucleic acid endonuclease according to claim 1, characterized in that, In step S2, the dephosphorylation treatment uses a dephosphorylase.

7. The method for rapidly obtaining the cleavage pattern of an unknown nucleic acid endonuclease according to claim 1, characterized in that, The T vector in step S3 was cloned using the TOPO cloning kit.

8. The method for rapidly obtaining the cleavage pattern of an unknown nucleic acid endonuclease according to claim 1, characterized in that, In step S3, the DNA fragment obtained in step S2 is ligated into the T vector using the TOPO cloning kit.

9. The method for rapidly obtaining the cleavage pattern of an unknown nucleic acid endonuclease according to claim 1, characterized in that, In step S4, the specific steps for screening positive clones are as follows: select the single clones from step S3, perform PCR amplification and detection using the amplification primers on the T vector, and obtain positive recombinants.

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