CN114836525A

CN114836525A - Method for rapidly obtaining unknown endonuclease cutting mode

Info

Publication number: CN114836525A
Application number: CN202110137807.XA
Authority: CN
Inventors: 朱斌; 成锐
Original assignee: Huazhong University of Science and Technology
Current assignee: Huazhong University of Science and Technology
Priority date: 2021-02-01
Filing date: 2021-02-01
Publication date: 2022-08-02

Abstract

The invention discloses a method for rapidly obtaining a cutting mode of unknown endonuclease, which comprises the steps of firstly processing a DNA fragment obtained by cutting the unknown endonuclease, then connecting the DNA fragment into a T carrier, sequencing primers respectively towards the direction of a connecting site of a connecting product to enable a sequencing result to cover the breaking position of a substrate DNA, and then, according to sequence information of a sequence of an exogenous DNA fragment (namely an original DNA fragment) and the adjacent T carrier boundary, carrying out sequence comparison analysis to accurately deduce the breaking position and the breaking mode of the substrate DNA fragment.

Description

Method for rapidly obtaining unknown endonuclease cutting mode

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a method for quickly obtaining an unknown endonuclease cutting mode.

Background

Nucleases are widely present in organisms, play an important role in nucleic acid metabolism, and are also widely used as important tool enzymes in molecular biology. The specificity and the mode of action of nucleases from different sources are different. Some nucleases act only on RNA, called ribonucleases (rnases), some can act only on DNA, called deoxyribonucleases (dnases), and some are less specific, acting both on RNA and DNA, and are therefore collectively referred to as nucleases (nucleases). Depending on the position of action of nucleases, nucleases can be further classified into exonucleases (exonuclease) and endonucleases (endonuclease). In the 70 s of the 20 th century, a class of endonucleases (restriction endonucleases, abbreviated as restriction enzymes) which can specifically recognize and hydrolyze specific nucleotide sequences on double-stranded DNA were discovered in bacteria in succession. When the foreign DNA invades into the bacteria, the restriction enzyme can hydrolyze and cut the foreign DNA into fragments, thereby limiting the expression of the foreign DNA in the bacterial cells, and the DNA of the bacteria is not hydrolyzed and protected because the DNA of the bacteria is modified by methylase at the specific nucleotide sequence. The research and application of restriction endonucleases are rapidly developed, more than 100 restriction endonucleases are purified, and many tool enzymes which are necessary for genetic engineering research are widely used for DNA molecular cloning and sequence determination. The endonuclease acts on a phosphodiester bond of double-stranded DNA, can recognize a specific base sequence and has a specific cutting site, and the determination of the cutting specificity of the unknown endonuclease has important significance for the research and the utilization of the endonuclease. Therefore, in order to better study the functional properties of unknown nucleases, it is important to accurately obtain their cleavage patterns.

In the prior art, chinese patent CN109207571A discloses a method for detecting endonuclease cut sites, which mainly utilizes billions of nucleic acid sequences on a chip to systematically detect the recognition cut sites and core sequence features of DNA by endonuclease in high throughput, and adopts twice sequencing to respectively obtain base sequences and normal double-stranded DNA, and meanwhile, according to the change of fluorescence signals before and after enzyme cutting, a biological information analysis method is used to obtain the recognition cut sites of endonuclease; through the research on the recognition and cutting sites of the endonuclease, the tendency of the recognition and cutting sites of the endonuclease can be detected, and the star activity of the endonuclease and the off-target effect of the endonuclease in the genome editing technology can be predicted in advance. Although the method has the advantage of high flux, the method has the defects of difficult technology, high cost, long time consumption, complex operation and the like.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a method for quickly obtaining the cutting mode of unknown endonuclease, which can accurately deduce the cutting position and the cutting mode of the unknown endonuclease, is used for determining the recognition site of the unknown nuclease, and has the advantages of simple and quick operation, accurate result, low cost, short time consumption and the like. Specifically, the following technique is used.

A method for rapidly obtaining an unknown endonuclease cleavage pattern comprises the following steps:

s1, cutting a DNA substrate by using unknown nuclease specificity to obtain a plurality of original DNA fragments, and performing gel cutting and recovery;

s2, carrying out terminal flattening and dephosphorylation treatment on the original DNA fragment recovered in the step S1;

s3, connecting the DNA fragment obtained in the step S2 into a T vector, and transforming the DNA fragment connected with the T vector into host bacteria;

s4, screening to obtain positive recombinants, and sequencing in the direction of the connection sites by using sequencing primers on the T carrier; deducing the breaking position and the breaking mode of the original DNA fragment according to the T vector boundary sequence and the DNA fragment sequence adjacent to the T vector boundary sequence, thus obtaining the cutting mode of the unknown endonuclease;

the sequencing primer is any segment of sequence on a T carrier, the distance between the first base of the sequencing primer and the connection site is 50-800bp, and the distance from the first base of the sequencing primer to the base of the connection site is started and stopped.

The principle of the method is as follows: (1) cutting a DNA substrate by adopting unknown nuclease to obtain a plurality of original DNA fragments after cutting, and performing gel cutting recovery, wherein the fracture position of the original DNA substrate is the cutting site of the unknown nuclease; (2) in order to obtain a cutting mode of unknown nuclease, carrying out terminal flattening and dephosphorylation treatment on a DNA fragment obtained by recovering the photoresist after cutting, and then respectively connecting into T carriers; (3) transforming the ligation product (T vector containing the treated DNA fragment) into host bacteria (such as Escherichia coli), picking out single clone, and performing PCR amplification detection by using amplification primers (such as M13F and M13R) on the T vector to screen out positive recombinants; (4) sequencing primers (such as M13F and M13R) on the T vector are used for sequencing in the direction of a connection site (namely the connection position of the T vector and the treated DNA fragment, namely the breaking position of the substrate DNA) respectively, and the breaking position and the cutting mode of the unknown endonuclease can be deduced through sequence comparison analysis according to the information of the DNA fragment sequence and the adjacent T vector sequence.

Preferably, the distance between the first base of the sequencing primer and the ligation site is 100-600 bp. The existing one-generation sequencing has inaccurate sequencing results for bases with distances of 0-50bp and above 800bp, and has certain errors. The invention limits the distance in the range, can avoid the region, ensures that the sequence information of the fracture position is in the range of 100-600bp of the sequencing result, and has more accurate result; the error caused by the defects of the current generation sequencing technology is avoided.

Preferably, the DNA substrate in step S1 has a length of 300-2000 bp. The DNA fragment used as the cleavage substrate can be selected according to the need, but it is generally preferable to use about 300-2000bp, and the too long or too short fragment is not good for the gel cutting recovery and ligation efficiency. Furthermore, the selected DNA fragment of the cutting substrate preferably only contains one cutting site, and 2 or more DNA fragments with obviously distinguishable sizes are obtained after cutting, so that the workload is reduced, the gel cutting recovery is effectively carried out, and the result is favorably obtained.

The technical scheme adopted by the invention is as follows: the sequencing primer is a sequence (such as M13F and M13R) on the T vector, when the length of the original DNA fragment obtained after cutting is 100-800bp, the sequences at two ends of the exogenous DNA fragment (namely the original DNA fragment) which is connected into the T vector can be obtained through one sequencing reaction; when the length of the original DNA fragment obtained after cutting is more than 800bp, only one sequence at one end of the exogenous DNA fragment connected into the T vector can be obtained through one sequencing reaction.

More preferably, the optimal length of the DNA substrate in step S1 is 600-1200 bp. In this case, the amplification primers for screening positive clones and the sequencing primers for detecting the cleavage site may be set to M13F and M13R, and the sequences at both ends of the foreign DNA fragment (i.e., the original DNA fragment) ligated to the T vector can be obtained by one sequencing reaction, thereby simplifying the procedure, simplifying the operation, and reducing the cost.

Preferably, after completion of steps S1 and S2, the DNA fragments recovered in step S1 and processed in step S2 are subjected to DNA purification processes, respectively. The DNA purification treatment adopts a DNA purification kit or an ethanol precipitation method.

Preferably, in step S2, the terminal-flattening process uses a rapid terminal-flattening kit. The 5' or 3' overhanging ends of the unlevel DNA are converted to blunt ends with 5' phosphate. The T4 DNA polymerase in the rapid terminal flattening kit has 3'→ 5' exonuclease activity and 5'→ 3' polymerase activity at the same time, so that the DNA terminal can be flattened; when the terminal is a 5' -end overhang, T4 DNA polymerase can fill in the terminal by its 5' → 3' DNA polymerase activity; when the end is a 3' overhang, T4 DNA polymerase can blunt the end using its 3' → 5' exodna activity. The rapid terminal flattening kit is a commercially available kit commonly used in the industry at present, such as the rapid terminal flattening kit produced by NEB (New England Biolabs, Inc.).

Preferably, in step S2, the dephosphorylation treatment employs a dephosphorylating enzyme.

Preferably, the T vector of step S3 employs a TOPO cloning kit.

Preferably, in step S3, the DNA fragment processed in step S2 is ligated into the T-vector using TOPO cloning kit.

Preferably, in step S4, the specific steps for screening positive clones are: the single clone in step S3 is picked up, PCR amplification detection is carried out by using the amplification primers (such as M13F and M13R) on the T vector, and a positive recombinant is obtained.

Compared with the prior art, the invention has the advantages that: the invention provides a brand new method for judging a cutting site and a cutting mode of unknown endonuclease, which is not found to be adopted by other domestic and foreign scientific research units at present. The method comprises the steps of processing a DNA fragment obtained by cutting unknown endonuclease, connecting the DNA fragment into a T vector, sequencing the DNA fragment in the direction of a connecting site of a connecting product by a sequencing primer respectively to enable a sequencing result to cover a breaking position of a substrate DNA, and then, according to sequence information of a sequence of an exogenous DNA fragment (namely an original DNA fragment) and an adjacent T vector boundary, accurately deducing the breaking position and the breaking mode of the substrate DNA fragment through sequence comparison analysis.

Drawings

FIG. 1 is a schematic flow chart I of the method for rapidly obtaining the cleavage pattern of an unknown endonuclease in example 1;

FIG. 2 is a schematic flow chart II of the method for detecting the cleavage pattern of an unknown endonuclease in example 1;

FIG. 3 shows the results of sequencing of the cleavage pattern of the unknown endonuclease in example 1.

Detailed Description

The technical solutions of the present invention will be described clearly and completely below, and it should be apparent that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

This example uses an endonuclease with an unknown cleavage pattern that cleaves a 955bp DNA fragment (DNA substrate) into 2 original 372bp and 583bp DNA fragments. In order to obtain the endonuclease cutting mode, firstly, cutting and recovering 2 original DNA fragments obtained by cutting, then carrying out terminal flattening and dephosphorylation treatment, then connecting into a T vector to obtain a connecting product, converting the connecting product (namely the T vector containing the treated DNA fragments) into escherichia coli, selecting a single clone, carrying out PCR amplification detection by using an amplification primer on the T vector to screen out positive recombinants, then selecting 10-20 positive recombinants, then sequencing by using a sequencing primer on the T vector to obtain sequence information of a joint part with the T vector, and then carrying out sequence comparison with a substrate DNA to accurately obtain the cutting site and the cutting mode of the endonuclease. The flow of this embodiment is shown in fig. 1 and 2, and the specific steps are as follows:

s1, specifically cutting a DNA substrate with the length of 955bp by using unknown nuclease to obtain 2 original DNA fragments with the lengths of 372bp and 583bp respectively, and cutting and recovering the gel;

s11, amplifying DNA fragments with known sequences by utilizing PCR, wherein the length of the DNA fragments is 955bp, and then purifying the amplified products, particularly, using a DNA purification kit (# T1030) produced by NEB (New England Biolabs, Inc. in the U.S.A.);

s12, cutting the DNA fragment of S11 serving as a reaction substrate by unknown nuclease to obtain 2 specific DNA fragments of 372bp and 583bp respectively, and performing gel cutting recovery and purification respectively;

s2, carrying out end flattening on the original DNA fragment recovered in the step S1 by adopting a rapid end flattening kit (# E1201) produced by NEB, carrying out reaction at room temperature for 15min, wherein the reaction system is shown in the following table 1; then, DNA purification was carried out by the same method as S11;

TABLE 1 terminal blunting and phosphorylation reaction System

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

Dephosphorylating with dephosphorylating enzyme (# M0525) produced by NEB at 37 deg.C for 10min, wherein the reaction system is shown in Table 2; then, DNA purification was carried out by the same method as S11;

TABLE 2 dephosphorylation reaction System

DNA	1pmol of DNAends
		10×CutSmart Buffer	2μL
Quick CIP	1μL
		Total volume	20μL

S3, connecting the DNA fragment obtained by processing in the step S2 into a T vector, and performing a connection reaction by adopting a second generation TOPO cloning kit (# C601-01) produced by Novozapine company, wherein the reaction system is shown in the following table 3, the reaction is performed at room temperature (20-37 ℃) for 5min to obtain a connection product, and the connection product (the DNA fragment connected with the T vector) is transformed into escherichia coli DH5 alpha;

TABLE 3 ligation reaction System

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

S4, selecting the monoclonal antibody in the step S3, carrying out PCR amplification by using an amplification primer (M13F/M13R primer pair which is commonly used in the field and has a known nucleotide sequence), then carrying out gel running detection, screening out a positive recon, sequencing in the direction of a connecting site by using a sequencing primer M13F, and obtaining the cutting position and the cutting mode of an original DNA fragment according to the known sequence and an adjacent plasmid sequence.

The sequencing primer in this example was chosen as M13F, and the sequencing results are shown in FIG. 3. In addition, it is well known to those skilled in the art that the sequencing primer can be any sequence on the T-vector, not only M13F or M13R, but also any sequence on the T-vector, and the technical scheme of the present invention can be realized by the sequencing primer as long as the sequencing result obtained by using the sequencing primer can cover the junction between the T-vector and the ligated DNA fragments and can ensure the accuracy of the base sequence near the junction.

When the ends of the original DNA fragments are leveled in step S2, different cutting methods may have different leveling methods, and when the ends protrude 5', the ends of the DNA may be filled up; 3' protruding the tail end, the tail end of the DNA can be flattened, and the flattened tail end has no change; these three cases can be confirmed based on the sequence information of the junction of the obtained T-vector and the foreign DNA. By picking different clones for sequencing, FIG. 3 exemplarily shows the sequencing result of the unknown endonuclease generated 5' overhanging ends of this example, and the bold sequences marked in FIG. 3 correspond to the sequences of the exogenous DNA ligated into the T-vector obtained by the sequencing reaction, respectively. From the above results, it can be obtained that the cleavage pattern of the unknown endonuclease used in this example is: positive strand 5' … AATAACC/CGGATATT … 3', negative strand 5' … AATATCC/GGGTTATT … 3' cleavage results in a DNA double strand break, with a single nucleotide overhang at the 5' end. When the method is used for detecting the unknown endonuclease cutting mode, the detection result is accurate, and the operation is simple and quick.

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

Claims

1. A method for rapidly obtaining an unknown endonuclease cleavage pattern is characterized by comprising the following steps:

the sequencing primer is any segment of sequence on the T carrier, and the distance between the first base of the sequencing primer and the connecting site is 50-800 bp.

2. The method for rapidly obtaining the cleavage pattern of unknown endonuclease as claimed in claim 1, wherein the distance between the first base of said sequencing primer and said ligation site is 100-600 bp.

3. The method for rapidly obtaining the cleavage pattern of unknown endonuclease as recited in claim 1, wherein the length of said DNA substrate in step S1 is 300-2000 bp.

4. The method for rapidly obtaining the cleavage pattern of unknown endonuclease as claimed in claim 3, wherein the optimal length of said DNA substrate in step S1 is 600-1200 bp.

5. The method of claim 1, wherein after steps S1 and S2 are completed, the DNA fragments recovered in step S1 and processed in step S2 are respectively subjected to DNA purification treatment.

6. The method for rapidly obtaining the cleavage pattern of an unknown endonuclease as recited in claim 1, wherein the end-flattening treatment in step S2 uses a rapid end-flattening kit.

7. The method for rapidly obtaining the cleavage pattern of an unknown endonuclease as claimed in claim 1, wherein the dephosphorylation treatment is performed by using a dephosphorylating enzyme in step S2.

8. The method for rapidly obtaining the cleavage pattern of an unknown endonuclease as claimed in claim 1, wherein the T vector of step S3 employs TOPO cloning kit.

9. The method of claim 1, wherein in step S3, the DNA fragment processed in step S2 is ligated into T-vector using TOPO cloning kit.

10. The method for rapidly obtaining the cleavage pattern of unknown endonuclease as claimed in claim 1, wherein the step of screening positive clones in step S4 comprises the following steps: and (5) selecting the monoclonal antibody in the step S3, and carrying out PCR amplification detection by using an amplification primer on the T vector to obtain a positive recombinant.