CN114836525A - Method for rapidly obtaining unknown endonuclease cutting mode - Google Patents

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

A rapid method for obtaining unknown endonuclease cleavage patterns

technical field

The invention belongs to the technical field of genetic engineering, in particular to a method for rapidly obtaining an unknown endonuclease cleavage pattern.

Background technique

Nucleases are widely present in organisms, they play an important role in nucleic acid metabolism, and are also widely used as important tool enzymes in molecular biology. Nucleases from different sources have different specificity and mode of action. Some nucleases can only act on RNA, called ribonuclease (RNase), and some can only act on DNA, called deoxyribonuclease (DNase). RNA can also act on DNA, so it is collectively called nuclease. Nucleases can be further divided into exonuclease and endonuclease according to the position of nuclease action. In the 1970s, a class of endonucleases was discovered in bacteria, which can specifically recognize and hydrolyze specific nucleotide sequences on double-stranded DNA, called restriction endonucleases (restriction endonucleases for short). enzymes). When exogenous DNA invades bacteria, restriction endonucleases can hydrolyze it into fragments, thus limiting the expression of exogenous DNA in bacterial cells, and the bacterial DNA itself is formazan at the specific nucleotide sequence. Modified by methylase, it will not be hydrolyzed and thus be protected. The research and application of restriction endonucleases have developed rapidly. There are more than 100 kinds of purified restriction endonucleases, many of which have become indispensable tool enzymes in genetic engineering research, and are widely used in DNA molecular cloning and sequence determination. Endonucleases act on the phosphodiester bonds of double-stranded DNA, can recognize specific base sequences, and have specific cleavage sites. The determination of the cleavage specificity of unknown endonucleases is of great significance for its research and utilization. Therefore, in order to better study the functional properties of unknown nucleases, it is crucial to accurately obtain their cleavage patterns.

In the prior art, Chinese patent CN109207571A discloses a method for detecting endonuclease cleavage sites, mainly using billions of nucleic acid sequences on a chip to systematically detect endonuclease pairs with high throughput. To identify the cleavage site and core sequence characteristics of DNA, two sequencings were used to obtain the base sequence and normal double-stranded DNA respectively. At the same time, according to the change of the fluorescence signal before and after the enzyme cleavage, the identification cleavage site of the endonuclease was obtained by using the method of bioinformatics analysis. Through the study of endonuclease recognition cleavage site, the tendency of endonuclease to recognize cleavage site can be detected, and the star activity of endonuclease and the off-target effect of endonuclease in genome editing technology can be predicted in advance . Although this method has the advantage of high throughput, it has disadvantages such as technical difficulty, high cost, long time, and complicated operation.

SUMMARY OF THE INVENTION

In view of the above deficiencies in the prior art, the present invention provides a method for rapidly obtaining the cleavage pattern of an unknown endonuclease, by which the cleavage position and the cleavage pattern of the unknown endonuclease can be accurately inferred, which is used for the unknown nuclease. It has the advantages of simple and rapid operation, accurate results, low cost, and short time-consuming. Specifically, it is realized by the following techniques.

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

S1. The DNA substrate is specifically cut with an unknown nuclease to obtain a number of original DNA fragments, which are recovered by cutting gel;

S2. blunt end and dephosphorylate the original DNA fragment recovered in step S1;

S3. connect the DNA fragment obtained by processing in step S2 into the T carrier, and transform the DNA fragment connected with the T carrier into the host bacteria;

S4. Screen to obtain positive recombinants, and use the sequencing primers on the T carrier to sequence the junction sites respectively; according to the border sequence of the T carrier and the sequence of the DNA fragment adjacent to it, infer the breaking position and breaking method of the original DNA fragment, That is, the cleavage pattern of the unknown endonuclease is obtained;

The sequencing primer is any sequence on the T carrier, and the distance between the first base of the sequencing primer and the ligation site is 50-800 bp, and the first base of the sequencing primer is The bases from the ligation site start and end.

The principle of the method is as follows: (1) using an unknown nuclease to cut the DNA substrate to obtain a number of original DNA fragments after cutting, and performing gel cutting to recover, and the breaking position of the original DNA substrate is the cutting site of the unknown nuclease; (2) In order to obtain the cleavage pattern of the unknown nuclease, the cut DNA fragments recovered from the cutting gel are subjected to end blunting and dephosphorylation treatment, and then ligated into the T vector respectively; The T carrier of the DNA fragment of the T carrier) is transformed into the host bacteria (such as Escherichia coli), pick a single clone, and use the amplification primers (such as M13F and M13R) on the T carrier to carry out PCR amplification detection to screen out positive recombinants; (4) Then use the sequencing primers (such as M13F and M13R) on the T carrier to sequence the junction site (that is, the junction position between the T carrier and the treated DNA fragment, that is, the breaking position of the substrate DNA), respectively. The information of the DNA fragment sequence and the adjacent T vector sequence can be deduced by the sequence alignment analysis, and the breaking position and cutting mode of the unknown endonuclease can be inferred.

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

Preferably, the length of the DNA substrate in step S1 is 300-2000 bp. The DNA fragment as the cutting substrate can be selected according to the needs, but generally about 300-2000 bp is suitable, and the fragment is too long or too short, which is not conducive to the recovery of cutting gel and the efficiency of ligation. Further, the selected cleavage substrate DNA fragment preferably contains only one cleavage site, and after cleavage, two or more DNA fragments with clearly distinguishable sizes should be obtained, so as to reduce the workload and effectively perform gel cutting recovery. conducive to obtaining results.

The above technical solution of the present invention is adopted: the sequencing primers are the sequences on the T carrier (such as M13F and M13R), and when the length of the original DNA fragment obtained after cutting is 100-800 bp, a sequencing reaction can be connected to the T carrier. The sequence at both ends of the exogenous DNA fragment (ie, the original DNA fragment); when the length of the original DNA fragment obtained after cutting is >800 bp, a sequencing reaction can only ensure that the sequence at one end of the exogenous DNA fragment ligated into the T vector is obtained.

More preferably, the optimal length of the DNA substrate in step S1 is 600-1200 bp. At this time, the amplification primers for screening positive clones and the sequencing primers for detecting the fragmentation position can be set to M13F and M13R, and a single sequencing reaction can obtain two exogenous DNA fragments (ie, original DNA fragments) linked to the T vector. end sequences to simplify procedures, make operations simpler, and reduce costs.

Preferably, after completing step S1 and step S2, DNA purification treatment is performed on the DNA fragment recovered in step S1 and the DNA fragment processed in step S2, respectively. DNA purification was carried out by DNA purification kit or ethanol precipitation.

Preferably, in step S2, the end flushing treatment adopts a rapid end flushing kit. Converts 5' or 3' overhangs of uneven DNA to blunt ends with 5' phosphates. The T4 DNA polymerase in the Rapid End Blunting Kit has both 3′→5′ exonuclease activity and 5′→3′ polymerase activity, which can blunt DNA ends; when the ends are 5′ protruding ends T4 DNA polymerase can use its 5'→3' DNA polymerase activity to fill in the end; when the end is overhanging the 3' end, T4 DNA polymerase can use its 3'→5' DNA exonuclease activity Flatten the ends. The rapid end blunting kit is currently a commercially available kit in the industry, such as the rapid end blunting kit (#E1201) produced by NEB (New England Biolabs, Inc., USA).

Preferably, in step S2, the dephosphorylation treatment adopts dephosphorylation enzyme.

Preferably, the T vector in step S3 adopts 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 of screening positive clones are: picking the single clone in step S3, and using amplification primers (eg M13F, M13R) on the T carrier to perform PCR amplification detection to obtain positive recombinants.

Compared with the prior art, the benefits of the present invention are: the present invention provides a brand-new method for judging the cleavage site and cleavage mode of an unknown endonuclease, and currently no other domestic and foreign scientific research units have adopted the method. . The DNA fragment obtained by cutting the unknown endonuclease is processed and connected to the T carrier, and the sequencing primers are sequenced in the direction of the ligation site of the ligated product, so that the sequencing result covers the breaking position of the substrate DNA, and then according to the exogenous DNA fragment. (i.e. the original DNA fragment) sequence and the sequence information of the adjacent T vector border, the sequence alignment and analysis can accurately infer the breaking position and breaking method of the substrate DNA fragment, so it can be used for the recognition site determination of unknown nucleases , has the advantages of simple and fast operation, accurate results, low cost, and short time-consuming.

Description of drawings

1 is a schematic flow chart I of the method for rapidly obtaining an unknown endonuclease cleavage pattern of Example 1;

2 is a schematic flow chart II of a method for detecting an unknown endonuclease cleavage pattern in Example 1;

FIG. 3 is the sequencing result of the cleavage pattern of the unknown endonuclease in Example 1. FIG.

Detailed ways

The technical solutions of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

Example 1

In this example, an endonuclease with an unknown cutting mode is used, which can cut a DNA fragment (DNA substrate) of 955 bp into two original DNA fragments of 372 bp and 583 bp. In order to obtain the endonuclease cleavage mode, the 2 original DNA fragments obtained by the cleavage are firstly recovered by cutting gel, then the ends are blunted and dephosphorylated, and then ligated into the T vector to obtain the ligation product, and then ligated. The product (that is, the T carrier containing the treated DNA fragment) was transformed into E. coli, and a single clone was picked, and the amplification primers on the T carrier were used for PCR amplification detection to screen out positive recombinants, and then select 10 -20 positive recombinants are sequenced with the sequencing primers on the T carrier to obtain the sequence information at the junction with the T carrier, and then the sequence alignment with the substrate DNA can accurately determine the cleavage site of the endonuclease and cutting patterns. The process flow of the present embodiment is shown in Figures 1 and 2, and the specific steps are as follows:

S1. The DNA substrate with a length of 955bp is specifically cut with an unknown nuclease to obtain 2 original DNA fragments with a length of 372bp and 583bp respectively, and the gel is cut and recovered;

S11, using PCR to amplify a DNA fragment with a known sequence, the length is 955bp, and then purify the amplified product, specifically using a DNA purification kit (#T1030) produced by NEB (New England Biolabs, Inc.) for DNA purification;

S12, the DNA fragment of S11 is used as a reaction substrate, and is cut with an unknown nuclease to obtain two specific DNA fragments of 372bp and 583bp respectively, which are respectively recovered and purified by gel cutting;

S2. The original DNA fragments recovered in step S1 were subjected to end-blunting using a rapid end-blunting kit (#E1201) produced by NEB, the reaction was carried out at room temperature, and the reaction time was 15min, and the reaction system was as shown in Table 1 below. shown; then the same method as S11 was used for DNA purification;

Table 1 End flushing and phosphorylation reaction system

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

Then use the dephosphorylation enzyme (#M0525) produced by NEB to carry out dephosphorylation treatment, and the reaction is carried out at 37° C., the reaction time is 10min, and the reaction system is shown in Table 2; then the same method as S11 is used to carry out DNA purification;

Table 2 Dephosphorylation reaction system

DNA 1pmol of DNAends 10×CutSmart Buffer 2μL Quick CIP 1μL total capacity 20μL

S3. Connect the DNA fragment obtained by the treatment in step S2 into the T carrier, and use the second-generation TOPO cloning kit (#C601-01) produced by Novozyme to carry out the ligation reaction. The reaction system is shown in Table 3 below, and the reaction is in Carry out at room temperature (20-37 ℃), the reaction time is 5min, obtain the ligation product, and transform the ligation product (DNA fragment connected with T vector) into Escherichia coli DH5α;

Table 3 ligation reaction system

5×Blunt Clonging Mix 1μL DNA fragment obtained in step S2 1μL Nuclease-free Water 3μL total capacity 5μL

S4. Pick the single clone in step S3, carry out PCR amplification with amplification primers (M13F/M13R primer pair commonly used in the art, the nucleotide sequence is known), run gel detection, screen out positive recombinants, and reuse The sequencing primer M13F is sequenced towards the ligation site, and the cutting position and cutting method of the original DNA fragment are obtained according to the known sequence and the adjacent plasmid sequence.

The sequencing primer in this example was selected as M13F, and the sequencing result is shown in FIG. 3 . In addition, it is well known to those skilled in the art that the sequencing primers are not only M13F and M13R, but can also be any sequence on the T carrier, as long as the sequencing results obtained by using the sequencing primers can cover the At the junction between the T vector and the connected DNA fragment, and the accuracy of the base sequence near the junction can be ensured, the sequencing primer can all realize the technical solution of the present invention.

When the original DNA fragment ends in step S2 are blunted, different cutting methods will also have different blunting methods. When the 5' overhang ends, the DNA ends will be filled; if the 3' overhang ends, the DNA ends will be blunted. The blunt ends have no change; these three cases can be confirmed according to the sequence information obtained at the junction between the T vector and the exogenous DNA. By picking different clones for sequencing, Fig. 3 exemplarily shows the sequencing results of the 5' overhangs generated by the unknown endonuclease of this example, and the bold sequences marked in Fig. 3 correspond to the concatenations obtained by the sequencing reaction respectively. The sequence on the foreign DNA into the T vector. According to 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', the cleavage results in DNA double-stranded Break, the break is a nucleotide overhang at the 5' end. When the method of the invention is used to detect the cleavage mode of the unknown endonuclease, the detection result is accurate, and the operation is simple and fast.

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

Claims (10)

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

Images