CN110079522A - A kind of application method of single layer molybdenum disulfide and the cutting method of DNA - Google Patents
A kind of application method of single layer molybdenum disulfide and the cutting method of DNA Download PDFInfo
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Abstract
The present invention provides the cutting method of a kind of application method of single layer molybdenum disulfide and DNA, the single layer molybdenum disulfide is used for cutting DNA.The cutting of DNA can be realized in the case where single layer molybdenum disulfide is used only and does not add any other auxiliary reagent in the cutting method of DNA provided by the invention at normal temperature.
Description
Technical field
The invention belongs to biomedicine technical field, it is related to a kind of application method of single layer molybdenum disulfide and the cutting of DNA
Method.
Background technique
Nucleic acid is the important large biological molecule of life entity, it is not only for the continuity of life, biological species hereditary capacity
Holding, growth and development, cell differentiation etc. play an important role, and and biomutation, such as tumour, hereditary disease, metabolic disease
Also closely related.So how effectively to manipulate nucleic acid molecules not only in terms of the disease treatments such as cancer, but also in biotechnology sheet
Body all has potential application.The initial stage fifties, biologist have found the limit to DNA by the behavior of infecting of bacteriophage
System and modification.The first restriction endonuclease Hind II is isolated within 1970 from haemophilus influenzae, is hewed out
The approach of one cutting DNA molecule.This fermentoid can identify certain special sequence on double chain DNA molecule, and be cut, shape
At the isolated fragment of certain length and sequence.But the research with interfering nucleic acid molecule was influenced with small organic molecule in recent years
The research interest of people is attracted.
Wherein, the artificial nuclease for simulating nucleic acid effectively cuts DNA DNA (Deoxyribonucleic
Acid research) achieves significant achievement；At present synthesized it is many can effectively cutting DNA Transition metal complexes
Object.For example, more classical Fe (Bleomycin), the metal complexs such as Mn (Porphyin) and Cu (Phenanthroline)
It can effective cutting DNA.The geometric configuration of metal complex plays vital work in the interaction process with DNA
With therefore carrying out reasonable design to metal complex and can not only change its steric configuration and electronic structure, and to grinding
The mechanism of action and biological function of they and DNA are studied carefully, to can identify the multi-functional of the conformation of DNA and the ability of sequence finding
Reagent plays an important role.In addition, going to explore with the repercussion study of small molecule transient metal complex and macromolecular DNA
Structure, mechanism of action and its function of macromolecular DNA, will be other research fields, as genetic chip, DNA biosensor,
The developmental research of DNA computer etc. provides most important theories basis.
Research finds the ability that there is the metal complex containing big planar structure ligand good and DNA to combine, because
This also has the cleavage activity of good DNA.But synthesis has the small molecular organic compounds of super plane structure, as gold
The ligand for belonging to ion forms the compound with larger flatness, when these compounds and DNA are acted on all must in illumination or
(document: Inorg.Chem.2007,46,11122-11132 can just occur under the action of other auxiliary reagents；
J.Phys.Chem.B 2010,114,5851-5861).CN102286458A discloses a kind of based on metal ion and oxidation stone
The DNA cutting method of black alkene, the patent is by preparation graphene oxide water solution, and by the graphene oxide and metal after sterilizing
Ion is added in DNA buffer, is reacted at normal temperature, realizes the cutting of DNA；Although the patent can not add any auxiliary
Reagent is irradiated without the use of light, but there is still a need for the cutting that could complete DNA is combined with metal ion for graphene oxide.
Current nano material such as C60, graphene oxide etc., the effect with DNA are needed in auxiliary reagent or illumination
The lower cutting being just able to achieve to DNA, therefore, it is necessary to develop the cutting method of new DNA a kind of.
Summary of the invention
The purpose of the present invention is to provide the cutting methods of a kind of application method of single layer molybdenum disulfide and DNA.The present invention
The cutting method of the DNA of offer is in the case where being used only single layer molybdenum disulfide and not adding any other auxiliary reagent, in room temperature
Under the cutting of DNA can be realized.
In order to achieve that object of the invention, the invention adopts the following technical scheme:
In a first aspect, the single layer molybdenum disulfide is used for the present invention provides a kind of application method of single layer molybdenum disulfide
Cutting DNA.
In the present invention, single layer molybdenum disulfide may be implemented in the case where not adding any other auxiliary reagent to DNA
Cutting.Compared with the small molecule DNA cutting agent of existing report, the plane and large specific surface area of two-dimension nano materials can be with
The dissection to DNA is improved, and then realizes the cutting to DNA.
Molybdenum disulfide can induce DNA by the generation of ROS (active oxygen) since its structure is two-dimension nano materials
Cutting, therefore, molybdenum disulfide can realize the cutting to DNA in the case where not adding any other auxiliary reagent.
Preferably, the application method are as follows: single layer molybdenum disulfide is mixed with DNA and is reacted, realizes the cutting of DNA.
Preferably, in the reaction system, the concentration of the single layer molybdenum disulfide is 1-500mg/L, such as 5mg/L, 10mg/
L, 50mg/L, 100mg/L, 150mg/L, 200mg/L, 250mg/L, 300mg/L, 350mg/L, 400mg/L, 450mg/L etc..
It is stronger to the dissection of DNA with the increase of concentration in the concentration range of 1-500mg/L, if being less than 1mg/
L, then due to the lower cutting being then difficult to realize to DNA of the concentration of molybdenum disulfide, if more than 500mg/L, it will cause to two sulphur
Change the waste of molybdenum.
Preferably, the size of the single layer molybdenum disulfide be 30-500nm, such as 40nm, 50nm, 60nm, 80nm,
100nm、120nm、150nm、170nm、200nm、220nm、250nm、280nm、300nm、320nm、340nm、380nm、
400nm, 420nm, 480nm etc..
In the range of 30-500nm, with the reduction of molybdenum disulfide size, the dissection of DNA is enhanced.
Preferably, the single layer molybdenum disulfide sterilizes before mixing with DNA.
Preferably, in the reaction system, pH value is 5 or more, such as 6,7,8,9,10,11,12,13 etc., further preferably
For 5-9.
With the increase of pH value, ROS yield increases, gradually increases to the dissection of DNA, if pH value is too small, ROS
Yield is too small, smaller to the dissection of DNA.
Preferably, the temperature of the reaction is 10-40 DEG C, such as 15 DEG C, 20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C etc..
Preferably, the time of the reaction be 0.5-30h, such as 1h, 2h, 5h, 10h, 12h, 15h, 18h, 20h, 25h,
28h etc..
Preferably, the DNA is added in the form of DNA buffer.
Preferably, the buffer is PBS buffer solution.
Preferably, in the reaction system, the concentration of the DNA be 0.01-0.02 μ g/ μ L, such as 0.012 μ g/ μ L,
0.013 μ g/ μ L, 0.014 μ g/ μ L, 0.015 μ g/ μ L, 0.016 μ g/ μ L, 0.018 μ g/ μ L etc..
Preferably, the preparation method of the single layer molybdenum disulfide is lithium ion graft process.
Single layer molybdenum disulfide of the invention can with the method for any available single layer molybdenum disulfide in the prior art into
Row, illustratively, lithium ion graft process.
Preferably, the DNA is selected from circular double stranded DNA.
As long as the DNA with double-strand, cyclic structure, the application method of single layer molybdenum disulfide provided by the invention can be with
Realize the cutting to DNA, cyclic DNA can be cut into linear DNA by the present invention.
As optimal technical scheme, the application method includes:
Single layer molybdenum disulfide having a size of 150-350nm is mixed with the PBS buffer solution of DNA and is carried out at 10-40 DEG C anti-
0.5-30h is answered, realizes the cutting of DNA.
Wherein, the concentration of single layer molybdenum disulfide is 1-500mg/L, and the concentration of DNA is 0.01-0.02 μ g/ μ L, reaction system
PH value be 5-9.
Second aspect, the present invention provides the cutting methods of DNA a kind of, utilize application method pair described in first aspect
DNA is cut.
Compared with the existing technology, the invention has the following advantages:
(1) in the present invention, single layer molybdenum disulfide may be implemented pair in the case where not adding any other auxiliary reagent
The cutting of DNA.
It (2) in the present invention, is within the scope of 1-500mg/L, having a size of 30-500nm model in the concentration of single layer molybdenum disulfide
It is stronger to the dissection of DNA with the increase of single layer molybdenum disulfide concentration, the reduction of size in enclosing；In the pH of reaction system
It is stronger to the dissection of DNA with the increase of pH value in the range of value is 5-9；Within the reaction time of 0.5-30h, reaction
Time is longer, stronger to the dissection of DNA.
Detailed description of the invention
Fig. 1 be embodiment 1, embodiment 5-7 DNA gel electrophoresis figure.
Fig. 2 be embodiment 1, embodiment 10-12 DNA gel electrophoresis figure.
Fig. 3 is the gel electrophoresis figure of the DNA of embodiment 1-4.
Fig. 4 be embodiment 1, embodiment 8-9 DNA gel electrophoresis figure.
Specific embodiment
The technical scheme of the invention is further explained by means of specific implementation.Those skilled in the art should be bright
, the described embodiments are merely helpful in understanding the present invention, should not be regarded as a specific limitation of the invention.
Embodiment 1
A kind of method of cutting DNA, the preparation method is as follows:
Single layer molybdenum disulfide having a size of 225nm is mixed with the PBS buffer solution of DNA and carries out reacting 18h at 25 DEG C, it is real
The cutting of existing DNA；
Wherein, the concentration of single layer molybdenum disulfide is 50mg/L, and the concentration of DNA is 0.015 μ g/ μ L, the pH value of reaction system
For 7, DNA pBR322.
Embodiment 2-4
Difference with embodiment 1 is only that in the present embodiment, the size of single layer molybdenum disulfide is in 175nm (embodiment
2), 300nm (embodiment 3), 325nm (embodiment 4).
Embodiment 5-7
Difference with embodiment 1 is only that in the present embodiment, the concentration of single layer molybdenum disulfide is 10mg/L (embodiment
5), 100mg/L (embodiment 6), 200mg/L (embodiment 7).
Embodiment 8-9
Difference with embodiment 1 is only that in the present embodiment, the pH value of reaction system is 5 (embodiments 8), 9 (implementations
Example 9).
Embodiment 10-12
Difference with embodiment 1 is only that in the present embodiment, the reaction time is 10h (embodiment 10), 15h (embodiment
11), 20h (embodiment 12).
Performance test
The DNA after DNA and cutting to embodiment offer is tested for the property, the method is as follows:
(1) it gel electrophoresis figure: is tested using day energy (Tannon) gel imager；
Fig. 1 be embodiment 1, embodiment 5-7 DNA gel electrophoresis figure, wherein Form I indicate cyclic DNA, Form
II indicates linear DNA, and swimming lane 1 be original DNA pBR322, swimming lane 2 be DNA, swimming lane 3 after the cutting of embodiment 1 is embodiment 5
DNA, swimming lane 4 after cutting be embodiment 6 cut after DNA, swimming lane 5 be embodiment 7 cut after DNA, can be seen by Fig. 1
Out, with the increase of molybdenum disulfide concentration, the dissection of DNA is gradually increased.
Fig. 2 be embodiment 1, embodiment 10-12 DNA gel electrophoresis figure, wherein swimming lane 1 be original DNA pBR322,
Swimming lane 2 be embodiment 1 cut after DNA, swimming lane 3 be embodiment 10 cut after DNA, swimming lane 4 be embodiment 11 cut after
DNA, swimming lane 5 are the DNA after embodiment 12 is cut, and as seen from Figure 2, with the increase in reaction time, are made to the cutting of DNA
With gradually increasing.
Fig. 3 is the gel electrophoresis figure of the DNA of embodiment 1-4, wherein swimming lane 1 is original DNA pBR322, swimming lane 2 is real
Apply example 4 cut after DNA, swimming lane 3 be embodiment 3 cut after DNA, swimming lane 4 be embodiment 1 cutting after DNA, swimming lane 5 be
DNA after embodiment 2 is cut with the reduction of molybdenum disulfide size, gradually increases the dissection of DNA as seen from Figure 3
By force.
Fig. 4 be embodiment 1, embodiment 8-9 DNA gel electrophoresis figure, wherein swimming lane 1-3 be respectively in pH 5,7,9
Under the conditions of original DNA pBR322, swimming lane 4 be embodiment 8 cut after DNA, swimming lane 5 be embodiment 1 cut after DNA, swimming
Road 6 is that the DNA after embodiment 9 is cut with the increase of pH, gradually increases the dissection of DNA as seen from Figure 4.
By Fig. 1-4 it is found that in the present invention, the cutting to DNA may be implemented in molybdenum disulfide.
The Applicant declares that the application method of the present invention is explained by the above embodiments single layer molybdenum disulfide of the invention and
The cutting method of DNA, but the invention is not limited to above-mentioned processing steps, that is, do not mean that the present invention must rely on above-mentioned technique
Step could be implemented.It should be clear to those skilled in the art, any improvement in the present invention, to selected by the present invention
The equivalence replacement of raw material and addition, the selection of concrete mode of auxiliary element etc. all fall within protection scope of the present invention and openly
Within the scope of.
Claims (10)
1. a kind of application method of single layer molybdenum disulfide, which is characterized in that the single layer molybdenum disulfide is used for cutting DNA.
2. application method according to claim 1, which is characterized in that the application method are as follows: by single layer molybdenum disulfide with
DNA mixing is reacted, and realizes the cutting of DNA.
3. application method according to claim 2, which is characterized in that in the reaction system, the single layer molybdenum disulfide
Concentration is 1-500mg/L；
Preferably, the size of the single layer molybdenum disulfide is 30-500nm；
Preferably, the single layer molybdenum disulfide sterilizes before mixing with DNA.
4. application method according to claim 2 or 3, which is characterized in that in the reaction system, pH value is 5 or more, into one
Walk preferred 5-9.
5. the application method according to any one of claim 2-4, which is characterized in that the temperature of the reaction is 10-
40℃；
Preferably, the time of the reaction is 0.5-30h.
6. the application method according to any one of claim 2-5, which is characterized in that the DNA is with DNA buffer
Form is added；
Preferably, the buffer is PBS buffer solution；
Preferably, in the reaction system, the concentration of the DNA is 0.01-0.02 μ g/ μ L.
7. application method described in any one of -6 according to claim 1, which is characterized in that the system of the single layer molybdenum disulfide
Preparation Method is lithium ion graft process.
8. application method described in any one of -7 according to claim 1, which is characterized in that the DNA is selected from cyclic annular double-strand
DNA。
9. application method described in any one of -8 according to claim 1, which is characterized in that the application method includes:
Single layer molybdenum disulfide having a size of 150-350nm is mixed with the PBS buffer solution of DNA and is reacted at 10-40 DEG C
0.5-30h realizes the cutting of DNA；
Wherein, the concentration of single layer molybdenum disulfide is 1-500mg/L, and the concentration of DNA is 0.01-0.02 μ g/ μ L, the pH of reaction system
Value is 5-9.
10. a kind of cutting method of DNA, which is characterized in that utilize application method pair described in any one of claim 1-9
DNA is cut.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117187238A (en) * | 2023-11-02 | 2023-12-08 | 清华大学深圳国际研究生院 | DNA shearing device and method |
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