CN116732076A - Closed linear DNA preparation method - Google Patents
Closed linear DNA preparation method Download PDFInfo
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- CN116732076A CN116732076A CN202310705124.9A CN202310705124A CN116732076A CN 116732076 A CN116732076 A CN 116732076A CN 202310705124 A CN202310705124 A CN 202310705124A CN 116732076 A CN116732076 A CN 116732076A
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- Prior art keywords
- linear dna
- closed linear
- plasmid
- target gene
- telomerase
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- 238000002360 preparation method Methods 0.000 title abstract description 12
- 108020004414 DNA Proteins 0.000 claims abstract description 47
- 239000013612 plasmid Substances 0.000 claims abstract description 37
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 24
- 108010017842 Telomerase Proteins 0.000 claims abstract description 20
- 230000001939 inductive effect Effects 0.000 claims abstract description 12
- 239000000203 mixture Substances 0.000 claims abstract description 10
- 239000000411 inducer Substances 0.000 claims abstract description 8
- 241000588724 Escherichia coli Species 0.000 claims abstract description 6
- 102000053602 DNA Human genes 0.000 claims abstract description 5
- 238000012258 culturing Methods 0.000 claims abstract description 4
- 238000000034 method Methods 0.000 claims description 14
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 claims description 9
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 claims description 9
- 241000894006 Bacteria Species 0.000 claims description 7
- 238000000746 purification Methods 0.000 claims description 5
- 125000000089 arabinosyl group Chemical group C1([C@@H](O)[C@H](O)[C@H](O)CO1)* 0.000 claims description 4
- 230000006698 induction Effects 0.000 claims description 4
- 230000001131 transforming effect Effects 0.000 claims description 3
- 238000006911 enzymatic reaction Methods 0.000 claims description 2
- 238000000926 separation method Methods 0.000 claims description 2
- 108090000790 Enzymes Proteins 0.000 abstract description 8
- 102000004190 Enzymes Human genes 0.000 abstract description 8
- 108020004707 nucleic acids Proteins 0.000 abstract description 2
- 102000039446 nucleic acids Human genes 0.000 abstract description 2
- 150000007523 nucleic acids Chemical class 0.000 abstract description 2
- 230000001737 promoting effect Effects 0.000 abstract description 2
- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 description 5
- 238000011282 treatment Methods 0.000 description 5
- 230000003321 amplification Effects 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- 238000001976 enzyme digestion Methods 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 238000011901 isothermal amplification Methods 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 230000000692 anti-sense effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000002939 deleterious effect Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/185—Escherichia
- C12R2001/19—Escherichia coli
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Crystallography & Structural Chemistry (AREA)
- Saccharide Compounds (AREA)
Abstract
The invention discloses a closed linear DNA preparation method, and relates to the technical field of nucleic acid. The preparation method comprises the following steps: s1: constructing a plasmid: the plasmid is a plasmid containing a target gene and a plasmid skeleton, wherein the plasmid skeleton contains an inducible promoter and a telomerase sequence, and both ends of the target gene are provided with telomerase recognition sites; s2: inducing and culturing to obtain a closed linear DNA molecule mixture; s3: and (3) purifying and recovering the mixture of the closed linear DNA molecules to obtain the closed linear DNA of the Target gene. The preparation method disclosed by the invention is used for promoting the expression telomerase of escherichia coli to directly cut plasmids through an inducer to form closed linear DNA molecules, so that the use of commercial enzymes can be reduced, the cost is greatly saved, and the yield is improved.
Description
Technical Field
The invention relates to the technical field of nucleic acid, in particular to a closed linear DNA preparation method.
Background
Traditional methods use commercial enzyme treatments in vitro to generate closed linear DNA are costly and limiting factors. For example, when the isothermal amplification enzyme is used for mass preparation, a large amount of amplification is needed, then telomerase enzyme digestion treatment is carried out, residues such as protein and the like are also needed to be removed through cleaning and purification, the commercial enzyme amount in the whole process is large, and the cost is high. In addition, there are many limitations, such as the amount of enzyme used, the treatment time, and residual problems, particularly in the enzyme treatment process.
The closed linear DNA molecules to which the present invention relates may be regarded as single stranded circular molecules, and in general, the closed linear DNA described herein is substantially fully complementary in sequence, although the structure may be tolerant of some minor variations or "wobble". When denatured, it is effectively a circular molecule comprising two strands, a forward strand (sense or forward strand) and a reverse strand (antisense or negative strand) adjacent to each other. This is in contrast to plasmid DNA, in which the complementary sequences (positive and negative strand) are located on different circular strands. Its unique structure means that it is easier to renature than a plasmid. Furthermore, the existing plasmid products are mainly crude DNA molecules, since they contain nucleotide sequences which are not required for a pharmaceutical function and may have deleterious effects on cells. The RNA products are not stable enough, and therefore, in the field of preparing DNA products such as DNA drugs, there is a need to provide improved methods for large amounts of amplified DNA. In particular, there is a need to provide improved methods for amplifying DNA in a specific form, such as closed linear DNA. Closed linear DNA molecules have particular utility in therapeutic applications due to their higher stability and safety compared to other forms of DNA.
Thus, there is a need for an improved method for mass production of closed linear DNA molecules at a lower cost.
Disclosure of Invention
The invention aims to provide a closed linear DNA preparation method which solves the following technical problems:
in the prior art, a large amount of isothermal amplification enzymes are needed for amplification in vitro to generate closed linear DNA, telomerase enzyme digestion treatment and cleaning purification are needed to remove residues such as protein, and the preparation method is complex and high in cost.
The aim of the invention can be achieved by the following technical scheme:
a closed linear DNA preparation method comprising the steps of:
s1: constructing a plasmid: the plasmid is a plasmid containing a target gene and a plasmid skeleton, wherein the plasmid skeleton contains an inducible promoter and a telomerase sequence, and both ends of the target gene are provided with telomerase recognition sites;
s2: induction culture: transforming the plasmid obtained in the step S1 into host bacteria, culturing overnight, and inducing by using an inducer matched with an inducible promoter, wherein the inducer induces the host bacteria to express the target gene and a telomerase sequence, and the telomerase cleaves the plasmid to obtain a closed linear DNA molecule mixture;
s3: and (3) purifying and recovering the mixture of the closed linear DNA molecules to obtain the closed linear DNA of the Target gene.
As a further aspect of the invention: the plasmid backbone contains a resistance gene.
As a further aspect of the invention: the inducible promoter is an arabinose promoter, and the inducer is arabinose.
As a further aspect of the invention: the host bacteria are escherichia coli.
As a further aspect of the invention: the purification method is any one of enzymatic reaction and chromatographic column separation.
As a further aspect of the invention: the mixture of closed linear DNA molecules comprises closed linear DNA of Target gene and plasmid skeleton.
The invention has the beneficial effects that:
the invention provides a preparation method of closed linear DNA, which is used for promoting the expression of telomerase in escherichia coli to directly cut plasmids by an inducer to form closed linear DNA molecules. The invention relates to the construction of a plasmid vector comprising a telomerase sequence and a telomerase recognition site, and also comprising an inducible promoter. The invention directly produces the closed linear DNA through escherichia coli, can reduce the use of commercial enzyme, greatly saves the cost and improves the yield.
Drawings
The invention is further described below with reference to the accompanying drawings.
FIG. 1 is a map of a P-Doggybone vector of the present invention;
FIG. 2 is a graph of gum prior to and after induction of arabinose according to the present invention;
FIG. 3 shows the Target gene closed linear DNA obtained after purification and recovery according to the present invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Referring to FIGS. 1-3, a closed linear DNA preparation method comprises the steps of:
s1: constructing a plasmid: the plasmid is a plasmid containing a target gene and a plasmid skeleton, wherein the plasmid skeleton contains an arabinose promoter, a telomerase sequence and a resistance gene, and both ends of the target gene are provided with telomerase recognition sites;
s2: induction culture: transforming the plasmid obtained in the step S1 into host bacteria, culturing overnight, and inducing the plasmid by using arabinose matched with an arabinose promoter, wherein the arabinose induces escherichia coli to express Target genes and telomerase sequences, and the telomerase cleaves the plasmid to obtain a closed linear DNA molecule mixture containing Target gene closed linear DNA and a plasmid skeleton;
s3: and (3) separating, purifying and recovering the closed linear DNA molecule mixture by using a chromatographic column to obtain the closed linear DNA of the Target gene.
The foregoing describes one embodiment of the present invention in detail, but the description is only a preferred embodiment of the present invention and should not be construed as limiting the scope of the invention. All equivalent changes and modifications within the scope of the present invention are intended to be covered by the present invention.
Claims (6)
1. A method for preparing closed linear DNA, comprising the steps of:
s1: constructing a plasmid: the plasmid is a plasmid containing a target gene and a plasmid skeleton, wherein the plasmid skeleton contains an inducible promoter and a telomerase sequence, and both ends of the target gene are provided with telomerase recognition sites;
s2: induction culture: transforming the plasmid obtained in the step S1 into host bacteria, culturing overnight, and inducing by using an inducer matched with an inducible promoter, wherein the inducer induces the host bacteria to express the target gene and a telomerase sequence, and the telomerase cleaves the plasmid to obtain a closed linear DNA molecule mixture;
s3: and (3) purifying and recovering the mixture of the closed linear DNA molecules to obtain the closed linear DNA of the Target gene.
2. The method for preparing closed linear DNA according to claim 1, wherein the plasmid backbone contains a resistance gene.
3. The method for preparing closed linear DNA according to claim 1, wherein the inducible promoter is an arabinose promoter and the inducer is arabinose.
4. The method for preparing closed linear DNA according to claim 1, wherein the host bacterium is Escherichia coli.
5. The method for preparing closed linear DNA according to claim 1, wherein the purification method is any one of enzymatic reaction and column separation.
6. The method of claim 1, wherein the mixture of closed linear DNA molecules comprises Target gene closed linear DNA and plasmid backbone.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310705124.9A CN116732076A (en) | 2023-06-14 | 2023-06-14 | Closed linear DNA preparation method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310705124.9A CN116732076A (en) | 2023-06-14 | 2023-06-14 | Closed linear DNA preparation method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116732076A true CN116732076A (en) | 2023-09-12 |
Family
ID=87902375
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310705124.9A Pending CN116732076A (en) | 2023-06-14 | 2023-06-14 | Closed linear DNA preparation method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116732076A (en) |
-
2023
- 2023-06-14 CN CN202310705124.9A patent/CN116732076A/en active Pending
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