CN113667727B - Method for obtaining linear plasmid DNA breaking position sequence information - Google Patents
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Abstract
The invention discloses a method for obtaining linear plasmid DNA breaking position sequence information, which relates to the technical field of molecular biology and comprises the following steps: s1, carrying out phosphorylation treatment on DNA fragments with known sequences; s2, carrying out end leveling and phosphorylation treatment on the linear plasmid to be detected; s3, carrying out blunt end ligation on the DNA fragment and the linear plasmid, and transforming the DNA fragment and the linear plasmid into host bacteria; s4, screening positive recombinants, sequencing the positive recombinants by using sequencing primers in the direction of a connecting site, and obtaining the breaking position and breaking mode of the linear plasmid according to the known sequence and the immediately adjacent plasmid sequence; the sequencing primer is any section of sequence on a connection product, and the distance between the sequencing primer and the connection site is 50-1000 bp. According to the sequence information of the connection position, the invention can accurately obtain the breaking position of the linear plasmid and the DNA sequence information near the breaking position, is used for determining the recognition site of unknown nuclease, and has the advantages of simple and quick operation, accurate result, low cost, short time consumption and the like.
Description
Technical Field
The invention relates to a method for obtaining sequence information of a linear plasmid DNA breaking position, which can effectively detect the breaking position of the linear plasmid DNA and belongs to the technical field of molecular biology.
Background
Plasmid DNA has three conformations, most of which generally exhibit supercoiled SC configuration, with both polynucleotide strands maintaining an intact circular structure, also known as covalently closed circular DNA (cccDNA); when only one of the two polynucleotide strands retains a complete circular structure, the other strand exhibits one or more gaps, also known as open loop DNA (ocDNA), i.e., OC configuration; when plasmid DNA is cleaved by a nuclease such as a restriction enzyme, double strand breaks occur to form linear molecules (IDNA), i.e., L-configuration. In agarose gel electrophoresis, the three different states of plasmid DNA phoresis rate is also different, supercoiled DNA phoresis rate is the fastest, linear plasmid DNA is the second slowest, and open-loop plasmid DNA is the slowest.
Plasmid DNA (e.g., pUC19 plasmid) is often used as a substrate for detecting nuclease activity of unknown proteins, and when treated with a protein having nuclease activity, it changes the electrophoresis characteristics of plasmid DNA, the supercoiled form of DNA decreases, and the linear and open-loop forms of DNA increases, so that the level of unknown nuclease activity can be verified based on the amounts of the linear and open-loop forms of DNA. However, the method can only verify the enzyme activity of unknown nuclease, and the sequence information of the recognition site cannot be obtained. Currently, the recognition site of nuclease is usually predicted by bioinformatics software, such as the poly zinc finger recognition sequence design software provided by www.zincfingers.org website, however, the software prediction is based on the amino acid sequence information of unknown nuclease, and the final function of the encoded protein cannot be directly determined according to the amino acid sequence information (which only determines the primary structure of the protein), so that the accuracy of the recognition site presumed based on the amino acid sequence analysis is difficult to determine; it still needs to be verified through experiments. Document (Hu Xiaoqian, tang Xin, J. Proc. Ind. Of Ministry of Hozhou, 2006,021 (003): 128-129, 123.) reports a spirulina intracellular nuclease, which cleaves substrate lambda-DNA and plasmid 8760 with spirulina intracellular nuclease, then electrophoretically detects the size of a plurality of DNA bands generated by the cleavage, and obtains recognition sites of the spirulina intracellular nuclease by comparing and analyzing electrophoresis pictures of the plurality of DNA bands with p 8760-related cleavage maps; however, this method is relatively complex; and when the substrate plasmid has a plurality of adjacent enzyme cutting sites, the size of DNA fragments generated by enzyme cutting is relatively close, and error results are easy to obtain. Therefore, in order to better study the functional properties of unknown nucleases, it is important to accurately obtain sequence information of recognition sites.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for obtaining the sequence information of the breaking position of the DNA of the linear plasmid, by using the method, the breaking position of the linear plasmid and the DNA sequence information near the breaking position can be accurately deduced, and the method is used for determining the recognition site of unknown nuclease and has the advantages of simple and rapid operation, accurate result, low cost, short time consumption and the like.
In order to achieve the above purpose, the invention adopts the following technical scheme:
a method for obtaining linear plasmid DNA fragmentation position sequence information, the flow chart of which is shown in figure 1, comprising the steps of:
s1, carrying out phosphorylation treatment on DNA fragments with known sequences;
s2, carrying out end leveling and phosphorylation treatment on the linear plasmid to be detected;
s3, carrying out blunt end ligation on the DNA fragment obtained in the step S1 and the linear plasmid obtained in the step S2 to obtain a ligation product, and converting the ligation product into host bacteria;
s4, screening positive recombinants, sequencing the positive recombinants towards the direction of a connecting site by using a sequencing primer, and obtaining the breaking position and the breaking mode of the linear plasmid according to the known sequence and the sequence of the plasmid in close proximity;
the sequencing primer is any section of sequence on a DNA fragment with a known sequence, the distance between the sequencing primer and the connecting site is 50-1000 bp, and the first base of the sequencing primer is located at a distance from the base of the connecting site.
The principle of the method is as follows: the ligation product (e.g., pUC19 plasmid) is cleaved with the unknown nuclease to yield a linear plasmid, the cleavage site of which is the recognition site of the unknown nuclease. To obtain sequence information of the cleavage site of the linear plasmid, purifying the linear plasmid to be detected, performing end leveling and phosphorylation, then performing blunt end ligation with a section of DNA fragment with known sequence after phosphorylation to obtain a ligation product-circular plasmid, then transferring the ligation product into host bacteria such as escherichia coli, picking up monoclonal, performing PCR amplification detection by using an amplification primer on the DNA fragment with known sequence to screen out positive recombinants, and then sequencing by using a sequencing primer in the direction of the ligation site (the ligation site of the linear plasmid and the DNA fragment, which is also the cleavage site of the linear plasmid), and determining the cleavage site and cleavage mode of the linear plasmid DNA by sequence comparison and analysis according to the sequence information of the known sequence and the immediately adjacent plasmid.
Preferably, the distance between the sequencing primer and the connecting site is 200-600 bp. Within the distance range, the sequence information of the breaking position can be ensured to be positioned within the range of 200-600 bp of the sequencing result, the result is more accurate, and errors caused by defects of the current generation of sequencing technology, such as inaccurate sequencing results of bases after 0-100 bp and 800bp in the generation of sequencing, can be avoided, and certain errors exist; therefore, the distance between the sequencing primer and the ligation site is set to 200 to 600bp, so that the region can be avoided.
Preferably, the length of the DNA fragment with the known sequence is 100-1000 bp. DNA fragments of known sequences can be selected as desired, but generally about 100 to 1000bp are preferred, and too long or too short fragments can reduce ligation efficiency. Further, a resistance gene (e.g., an ampicillin resistance gene) different from the linear plasmid to be detected may be added to a DNA fragment of a known sequence to increase the recombination positive rate.
The technical scheme is adopted: when the length of the DNA fragment with the known sequence is 100-600 bp, the sequencing primer can be the same as or different from the amplification primer used for screening the positive clones; and when the length of the DNA fragment with the known sequence is 600-1000 bp, the sequencing primer is positioned at the downstream of the amplification primer; thereby ensuring that the distance between the sequencing primer and the connecting site is 200-600 bp.
Further preferably, the length of the DNA fragment of the known sequence is 400 to 600bp. In this case, the amplification primer for screening positive clones and the sequencing primer for detecting the cleavage site can be set to be the same pair, thereby simplifying the procedure, simplifying the operation, and reducing the cost.
Preferably, DNA purification is performed after the treatment in steps S1 to S2.
Preferably, the phosphorylation in step S1 is performed using a polynucleotide kinase.
Preferably, the end-blunting and phosphorylating treatment in step S2 uses a rapid end-blunting kit to convert the 5' or 3' overhanging ends of non-blunt DNA into blunt ends with 5' phosphate. The rapid end leveling kit is an enzyme mixed solution, and comprises DNA polymerase and polynucleotide kinase, wherein the T4 DNA polymerase has 3 '. Fwdarw.5' exonuclease activity and 5 '. Fwdarw.3' polymerase activity at the same time, so that the DNA end can be leveled; when the terminal is a 5' -terminal protruding terminal, the T4 DNA polymerase can use its 5 '. Fwdarw.3 ' DNA polymerase activity to fill in the terminal; when the terminal is a 3' -terminal protruding terminal, the T4 DNA polymerase can use its 3 '. Fwdarw.5 ' DNA exonuclease activity to flatten the terminal; t4 polynucleotide kinase phosphorylates the 5' end of blunt-ended DNA for ligation reactions.
Preferably, the blunt end ligation in step S3 employs a DNA ligase.
Preferably, the specific steps of screening positive clones in step S4 are as follows: and (3) selecting a monoclonal, and carrying out PCR amplification detection by using an amplification primer on a DNA fragment with a known sequence to obtain a positive recombinant.
The invention has the advantages that: sequencing is carried out by utilizing sequencing primers to the connection site direction of the connection product respectively, so that the sequencing result covers the breaking position of the linear plasmid, and then the breaking position, the breaking mode and DNA sequence information near the breaking position of the linear plasmid can be accurately deduced through sequence comparison analysis according to the known sequence and the sequence information of the immediately adjacent plasmid, thereby being applicable to the determination of the recognition site of unknown nuclease and having the advantages of simple and rapid operation, accurate result, low cost, short time consumption and the like.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly described below, and it is apparent that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a flow chart of a method for obtaining linear plasmid DNA fragmentation position sequence information;
FIG. 2 is a flow chart of a method for detecting pUC19 linear plasmid DNA fragmentation position information according to an embodiment;
FIG. 3 shows three cleavage patterns of the linear pUC19 plasmid detected in the examples; FIG. 3-A shows pUC19 plasmid cleavage to generate 5' overhanging ends; FIG. 3-B shows pUC19 plasmid cleavage to generate 3' overhanging ends; FIG. 3-C shows pUC19 plasmid cleavage to generate blunt ends.
FIG. 4 shows the sequencing results of pUC19 plasmid cleavage in the examples to generate three terminal cleavage modes; the bolded sequences marked in the figures correspond to the sequences on the fragmented plasmids obtained by the sequencing reaction, respectively, and the italic sequences marked in the figures are the sequences with the filled ends.
Detailed Description
The following description of the embodiments of the present invention will be made more apparent and fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments of the invention are shown. All other embodiments, which can be made by one of ordinary skill in the art without undue burden on the person of ordinary skill in the art based on embodiments of the present invention, are within the scope of the present invention.
Examples
In this example, a nuclease with unknown function is used, which can treat supercoiled plasmid DNA into linear and open-loop plasmids, to obtain the sequence information of the breaking position of the linear plasmid DNA, firstly, the linear plasmid is cut and recovered, then the end is leveled and phosphorylated, and then the linear plasmid is connected with a section of DNA fragment with known sequence after being phosphorylated to obtain a connection product, then the connection product is transformed into E.coli, monoclonal is picked up, amplified and detected by amplification primers on the DNA fragment with known sequence to screen out positive recombinants, then 10-20 positive recombinants are selected, and sequencing primers are used to sequence the two ends respectively, so as to obtain the sequence information of the breaking position of the linear plasmid, and then the sequence information is compared with the plasmid, so that the sequence information of the breaking position of the linear plasmid can be accurately obtained. The flow of the embodiment is shown in fig. 2, and the specific steps are as follows:
s1, carrying out phosphorylation treatment on DNA fragments with known sequences;
s11.PCR amplification of DNA fragments of known sequence: selecting a section of DNA fragment with a known sequence and length of 600bp, adopting amplification primers F1 and R1 to perform PCR amplification to obtain an amplification product, and purifying the amplification product, wherein a DNA purification kit (#T1030) produced by NEB is specifically used for DNA purification;
s12, phosphorylation treatment of DNA fragments with known sequences: the T4 polynucleotide kinase (#M0201) produced by NEB was used for phosphorylation, and the reaction system was as shown in Table 1:
table 1T4 Polynucleotide kinase phosphorylation reaction System
The phosphorylation reaction is carried out at 37 ℃ for 30min, and then DNA purification is carried out;
s2, carrying out end leveling and phosphorylation treatment on the linear plasmid to be detected; carrying out electrophoresis gel running on pUC19 plasmid reacted with unknown nuclease, then cutting gel, recovering, and then carrying out DNA purification; then, a rapid end flush kit (#E1201) produced by NEB was used for end flush and phosphorylation treatment, and the reaction system was as shown in Table 2:
TABLE 2 end-blunting and phosphorylation reaction System
The reaction was carried out at room temperature for 15min, and then DNA purification was carried out.
S3, carrying out blunt end ligation on the DNA fragment obtained in the step S1 and the linear plasmid obtained in the step S2 through high-concentration T4 DNA ligase (#M0202) produced by NEB, carrying out a reaction at room temperature for 10min to obtain a ligation product-circular plasmid, and then converting the ligation product into escherichia coli DH5 alpha;
TABLE 3 Flat end ligation reaction System
S4, selecting the monoclonal in the step S3, performing PCR amplification by using an amplification primer F1 and an amplification primer R1, performing gel running detection, screening out positive recombinants, sequencing by using a sequencing primer in the direction of a connecting site, and obtaining the breaking position and the breaking mode of the linear plasmid according to the known sequence and the plasmid sequence in the close vicinity; in this example, the sequencing primer was selected as the F1 and R1 primer pair, and the sequencing result is shown in FIG. 3. It should be understood by those skilled in the art that the sequencing primer may be any sequence on a DNA fragment of a known sequence, so long as the sequencing result obtained by using the sequencing primer can cover the cleavage site of the linear plasmid and ensure the accuracy of the base sequence near the cleavage site, the sequencing primer can implement the technical scheme of the present invention, that is, the position and sequence information of the sequencing primer in the present embodiment cannot be used as a limitation on the protection scope of the present invention.
As can be seen from FIG. 3, the unknown nucleases used in this example have no specificity in their recognition sites and may generate three cleavage events, namely, a 5 'overhang, a 3' overhang and a blunt end. When the tail ends are leveled, different fracture modes can be leveled, and when the 5' protrudes out of the tail ends, linear plasmid DNA can be supplemented; 3' protruding end, linear plasmid DNA will be flattened, the flat end will not change; these three cases correspond to A-C in FIG. 3, respectively, and are confirmed based on the sequence information of the junction of the obtained linear plasmid with the known sequence. As a result of sequencing by picking different clones, it was found that three linear pUC19 plasmids were detected in total in a way that resulted in 5 'overhanging ends, 3' overhanging ends and blunt ends for cleavage, respectively, as shown in FIG. 3; in FIG. 3-A, the 5' overhang creates a repetitive sequence due to the filling-in of both end boundaries; in FIG. 3-B, the 3' overhanging end would generate a sequence deletion due to flattening of the two end boundaries; the blunt-ended sequence in FIG. 3-C was unchanged. FIG. 4 is an exemplary illustration of the sequencing results of pUC19 plasmid cleavage resulting in three terminal cleavage modes. From the above results, three cleavage sites of the unknown nucleases used in this example were obtained, respectively:
1) The positive strand 5' … GCCA/GTGA … 3', the negative strand 5' … ATTC/ACTG … 3', the cleavage results in a DNA double-strand break with 5' two nucleotide overhangs (see FIG. 4-A);
2) The positive strand 5' … AGCG/GGTG … ' and the negative strand 5' … ACCC/GCTG … 3' are cut to cause DNA double strand break, the break is 3' one nucleotide protrusion (see figure 4-B);
3) The positive strand 5'… GAAA/CCTG … 3', the negative strand 5'… CAGG/TTTC … 3', the cleavage results in a DNA double-strand break, the break is flush and no protrusion (see FIG. 4-C).
When the method is used for detecting the sequence information of the linear plasmid DNA breaking position, the detection result is accurate, and different breaking modes can be detected by the method, so that the operation is simple and quick.
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, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (9)
1. A method for obtaining linear plasmid DNA fragmentation site sequence information, comprising the steps of:
s1, carrying out phosphorylation treatment on DNA fragments with known sequences;
s2, carrying out end leveling and phosphorylation treatment on the linear plasmid to be detected;
s3, carrying out blunt end ligation on the DNA fragment obtained in the step S1 and the linear plasmid obtained in the step S2 to obtain a ligation product, and converting the ligation product into host bacteria;
s4, screening positive recombinants, sequencing the positive recombinants towards the direction of a connecting site by using a sequencing primer, and obtaining the breaking position and the breaking mode of the linear plasmid according to the DNA fragment sequence of a known sequence and the sequence of the plasmid adjacent to the DNA fragment sequence;
the sequencing primer is designed aiming at the DNA fragment sequence with a known sequence, and the distance between the first base at the 5' end of the sequencing primer and the connecting site at the downstream of the primer is 200-600 bp; the ligation site is the ligation site of the linear plasmid and the DNA fragment, and the ligation site is the cleavage site of the linear plasmid.
2. The method for obtaining linear plasmid DNA fragmentation site sequence information according to claim 1, wherein the length of the DNA fragment of the known sequence is 100-1000 bp.
3. The method for obtaining linear plasmid DNA fragmentation site sequence information according to claim 2, wherein the length of the DNA fragment of the known sequence is 400-600 bp.
4. The method according to claim 1, wherein the DNA fragment of known sequence contains a resistance gene different from the linear plasmid to be detected.
5. The method for obtaining linear plasmid DNA cleavage site sequence information according to claim 1, wherein the DNA purification is performed after the treatment in steps S1 to S2.
6. The method of claim 1, wherein the phosphorylation treatment in step S1 is performed using T4 polynucleotide kinase.
7. The method of claim 1, wherein the end-blunting and phosphorylating treatment in step S2 is performed using a rapid end-blunting kit; the rapid end leveling kit is an enzyme mixed solution, and comprises T4 DNA polymerase and T4 polynucleotide kinase, wherein the T4 DNA polymerase has 3' -exonuclease activity and 5' -3 ' -polymerase activity at the same time, so that the DNA ends are leveled; when the terminal is a 5' -terminal protruding terminal, the T4 DNA polymerase can use the activity of the 5' -3 ' -DNA polymerase to fill in the terminal; when the terminal is 3' -terminal protruding terminal, the T4 DNA polymerase can use 3' -5 ' -DNA exonuclease activity to flatten the terminal; t4 polynucleotide kinase phosphorylates the 5' -end of blunt-ended DNA for ligation reactions.
8. The method of claim 1, wherein the blunt-ended ligation is performed using T4 DNA ligase in step S3.
9. The method for obtaining linear plasmid DNA cleavage site sequence information according to claim 1, wherein the specific step of screening the positive recombinants in step S4 is as follows: and (3) selecting monoclonal, performing PCR amplification detection by using an amplification primer designed for a DNA fragment sequence with a known sequence, and screening out positive recombinants.
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