CN108998442B - Method for shearing DNA molecule and application thereof - Google Patents
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Abstract
The invention discloses a method for shearing DNA molecules and application thereof, belonging to the technical field of genetic engineering. Mixing chiral semiconductor nanoparticles and DNA molecules, incubating, and irradiating the incubated mixture of the chiral semiconductor nanoparticles and the DNA molecules under circularly polarized light to obtain sheared DNA molecules; the method of the invention can quickly obtain a large number of DNA fragments with uniform structures and high specificity on sequences and sites, and has great application potential in the field of genetic engineering.
Description
Technical Field
The invention relates to a method for shearing DNA molecules and application thereof, belonging to the technical field of genetic engineering.
Background
Obtaining DNA fragments is a key step for techniques such as gene sequencing, gene analysis, gene editing, and the like. For gene sequencing, the method can effectively obtain a target fragment; for gene analysis, it can obtain single gene information; for gene editing, it can selectively extract or replicate gene sequences. Therefore, the shearing of DNA molecules is an indispensable pretreatment step in the field of genetic engineering.
With the continuous and deep research in the gene field, the demand for obtaining DNA fragments with uniform structure and high specificity on sequence and site is becoming greater and greater.
At present, the following methods are commonly used for shearing DNA molecules: DNA restriction, sonication, hydrodynamic shearing, etc., which are currently used for the generation of DNA fragments, but restriction endonuclease still requires a specific biological enzyme and has the drawback of high requirements for pH and temperature; the ultrasonic degradation method and the hydrodynamic shearing method have the defect of no specificity to a target sequence.
Therefore, the above methods cannot well obtain DNA fragments with uniform structure and high specificity on sequence and site, and a method for obtaining DNA fragments with uniform structure and high specificity on sequence and site is urgently needed to meet the needs of gene field research.
Disclosure of Invention
The chiral semiconductor nano particle consists of IV, II-VI, IV-VI or III-V elements and is commonly used for sensing detection, imaging, catalysis and the like; circularly polarized light is the locus traced by the end of the electric field or light vector of light in a plane perpendicular to the direction of propagation and is commonly used for characterization of chiral active substances.
To solve the above problems, the present invention provides a method for cleaving a DNA molecule. The method combines chiral semiconductor nanoparticles with circularly polarized light, and utilizes the principle that the chiral nanoparticles have special configuration and are easy to generate light-induced oxidation under light, the chiral semiconductor nanoparticles and DNA molecules are mixed and incubated, and then the incubated chiral semiconductor nanoparticles and DNA molecule mixture is irradiated under circularly polarized light, so that sheared DNA molecules are obtained; by using the method, a large number of DNA fragments which have uniform structures and high specificity on sequences and sites can be quickly obtained, and the method has huge application potential in the field of genetic engineering.
The technical scheme of the invention is as follows:
the invention provides a method for shearing DNA molecules, which comprises the steps of mixing chiral semiconductor nanoparticles with DNA molecules and then incubating, and then irradiating the incubated mixture of the chiral semiconductor nanoparticles and the DNA molecules under circularly polarized light to obtain the sheared DNA molecules.
In one embodiment of the invention, the method is to dissolve the DNA molecule in PBS buffer solution to obtain DNA molecule solution; adding the chiral semiconductor nano particles into a DNA molecular solution for mixing to obtain a mixed solution; standing the mixed solution for incubation to obtain an incubated mixed solution; and irradiating the incubated mixed solution under circularly polarized light to obtain the sheared DNA molecules.
In one embodiment of the present invention, the concentration of the PBS buffer is 0.01 mol/L.
In one embodiment of the invention, the PBS buffer has a pH of 7.4.
In one embodiment of the present invention, the concentration of the DNA molecules in the DNA molecule solution is 0.5 to 1.5. mu. mol/L.
In one embodiment of the present invention, the concentration of the DNA molecules in the DNA molecule solution is 1. mu. mol/L.
In one embodiment of the present invention, the chiral semiconductor nanoparticles are synthesized with D-cysteine or L-cysteine as chiral ligands.
In one embodiment of the invention, the molar ratio of the chiral semiconductor nanoparticles to the DNA molecules is 45-55: 1.
In one embodiment of the invention, the molar ratio of the chiral semiconductor nanoparticles to the DNA molecules is 50: 1.
In one embodiment of the invention, the time of standing is 0.5 to 1.5 hours.
In one embodiment of the invention, the time of standing is 1 h.
In one embodiment of the present invention, the irradiation is performed by adding the incubated mixture to a cuvette with a stopper and then irradiating the mixture under circularly polarized light.
In one embodiment of the invention, the irradiation time is 1 to 3 hours.
In one embodiment of the present invention, the irradiation time is 2 h.
In one embodiment of the present invention, the circularly polarized light is left circularly polarized light and/or right circularly polarized light.
In one embodiment of the present invention, the light source of the circularly polarized light is a laser.
In one embodiment of the present invention, the light source of the circularly polarized light is a 405nm laser.
In one embodiment of the invention, the circularly polarized light is obtained by passing a light source through quarter-glass slides of different angles.
The invention provides a DNA molecule fragment obtained by shearing through the method for shearing the DNA molecule.
The invention provides application of the DNA molecule shearing method in genetic engineering.
Has the advantages that:
(1) by using the method of the invention, a large number of DNA fragments which have uniform structures and high specificity on a sequence (GAT-ATC) and a locus (between T-A) can be rapidly obtained, and the method has great application potential in the field of genetic engineering;
(2) the chiral nanoparticles adopted by the invention are insensitive to temperature and pH, and can be applied to DNA shearing under various complex conditions;
(3) the chiral nanoparticles adopted by the invention have high specificity and biocompatibility, can generate DNA specific shear in living cells and tumors, and have potential application in clinical disease treatment.
Drawings
FIG. 1 is a transmission electron micrograph of chiral semiconductor nanoparticles;
FIG. 2 is a circular dichroism spectrum of a chiral semiconductor nanoparticle;
FIG. 3 electrophoretograms of a mixture of Salmonon DNA and L-Cys CdTe before and after 2h of irradiation with right circularly polarized light;
FIG. 4 electrophoretogram of mixture of Salmonon DNA and D-Cys CdTe before and after 2h of left circular polarized light irradiation;
FIG. 5 electrophorograms before and after 2h of a mixture of non-specific Salmonon DNA sequence and L-Cys CdTe under right circular polarized light irradiation;
FIG. 6 electrophoretogram before and after irradiating mixture of Salmonon DNA and L-GSH CdTe under circularly polarized light for 2 h;
FIG. 7 electrophoretograms of a mixture of Salmonon DNA and L-GSH CdTe before and after (a)0 deg.C (b)50 deg.C and exposure to circularly polarized light for 2 h;
FIG. 8 electrophoretogram of mixture of Salmonon DNA and L-GSH CdTe in (a) pH 6(b) pH 8 solution and before and after irradiating with circularly polarized light for 2 h.
Detailed Description
The present invention will be further illustrated below by taking Salmonon DNA as an example and combining specific examples and comparative examples.
The invention relates to a characterization method which comprises the following steps:
the DNA shearing effect characterization method comprises the following steps: 20uL of the sheared DNA product was taken and mixed with a loading buffer (Invitrogen). Then added rapidly to a 2.5% agarose gel prepared beforehand only. After the sample settled to the bottom of the gel well, the gel was placed in running buffer (Invitrogen)TM) The operation was carried out at 110V for 30 min. Finally, the DNA bands were detected on a gel imager.
An electron microscope characterization method comprises the following steps: the liquid sample was dropped on the carbon-supported membrane, and after 5min the sample was removed with absorbent paper. The copper mesh was then placed in a transmission electron microscope sample holder and the sample was observed at 220KV accelerating voltage.
Example 1: synthesis of precursors
Under the protection of nitrogen, 4mL of 0.5M H2SO4To 0.05g of Al2Te3And obtaining precursor gas in the solution.
Example 2: synthesis of chiral semiconductor nanoparticles
0.985g of Cd (ClO)4)2·6H2Adding O and 3mL of 1M D-type cysteine into 125mL of water, introducing the precursor gas obtained in example 1, adjusting the pH to 12 by adopting NaOH, adopting nitrogen as carrier gas for the precursor gas, controlling the flow rate to be 100mL/min, heating the solution to 110 ℃ while stirring, keeping the temperature for 8h, adding isopropanol into the obtained nanoparticle solution according to the volume ratio of 1:1, and centrifuging the mixture at 10000rpm for 5 min. The pellet was then resuspended in PBS buffer (0.01M, pH 7.4.4) to yield chiral semiconductor nanoparticles. (FIG. 1 is a transmission electron micrograph of a chiral semiconductor nanoparticle, and FIG. 2 is a circular dichroism of the chiral semiconductor nanoparticleSpectrogram)
Example 3: synthesis of chiral semiconductor nanoparticles
0.985g of Cd (ClO)4)2·6H2O and 3mL of 1M L-cysteine are added into 125mL of water, the precursor gas obtained in the embodiment 1 is introduced, the pH value is adjusted to 12 by adopting NaOH, nitrogen is adopted as carrier gas for the precursor gas, the flow rate is controlled to be 100mL/min, the solution is heated to 110 ℃ while being stirred, the temperature is kept for 8 hours, then isopropanol is added into the obtained nanoparticle solution according to the volume ratio of 1:1, and then the mixture is centrifuged for 5 minutes at 10000 rpm. The pellet was then resuspended in PBS buffer (0.01M, pH 7.4.4) to yield chiral semiconductor nanoparticles. (FIG. 1 is a transmission electron microscope image of chiral semiconductor nanoparticles, and FIG. 2 is a circular dichroism spectrum of chiral semiconductor nanoparticles)
Example 4: incubation of chiral semiconductor nanoparticles with DNA
50. mu.L of a 1. mu.M solution of Salmon sperm DNA (Salmon DNA) having the nucleotide sequence of SEQ ID NO.1 (obtained by dissolving Salmon DNA in 0.01M, pH 7.4.4 concentration PBS buffer) was thoroughly mixed with the chiral semiconductor nanoparticles obtained in examples 2 and 3 at a molar ratio of 50:1, respectively, and then allowed to stand for 1 hour to obtain a mixture of the incubated chiral semiconductor nanoparticles and DNA.
Example 5: irradiation of chiral semiconductor nanoparticles with DNA
100. mu.L of the mixture obtained in example 4 was taken and put into a cuvette with a stopper, and then the mixture was irradiated with left and right circularly polarized lights obtained by adjusting quarter slides at different angles for 2 hours using a laser of 405nm as a light source, respectively.
The irradiated mixture was centrifuged at 8000rpm for 10min and then characterized.
The characterization results are shown in FIGS. 3-4, from which it can be seen that: after mixing the salmon DNA with the chiral semiconductor nano-particles, after irradiating with circularly polarized light for 2 hours, the T and A basic groups in the GATATC specific fragment on the DNA sequence are broken, and the original length of the salmon DNA (the nucleotide sequence is SEQ ID NO.1) is 1839bp, and the cut lengths are 1083bp and 756bp respectively (the nucleotide sequences of the cut DNA fragments are SEQ ID NO.2 and SEQ ID NO.3 respectively).
(since there is little difference in the final results obtained by using D-cysteine and L-cysteine, only the results for D-cysteine are shown here)
Example 6: effect of temperature on the results of DNA cleavage
100 μ L of the mixture obtained in example 4 was taken and put into a cuvette with a stopper, and the cuvette was placed in an environment of 0 degrees and 50 degrees, respectively, and then the mixture was irradiated with left and right circularly polarized lights obtained by adjusting quarter slides at different angles for 2 hours, respectively, using a laser of 405nm as a light source. The irradiated mixture was centrifuged at 8000rpm for 10min and then characterized.
The characterization results are shown in FIG. 7, from which it can be seen that: after mixing the salmon DNA and the chiral semiconductor nano-particles, irradiating the mixture for 2 hours at 0 ℃ and 50 ℃ respectively by adopting circularly polarized light, and then breaking the T and A basic groups in a GATATC specific fragment on the DNA sequence, wherein the original length of the salmon DNA (the nucleotide sequence is SEQ ID NO.1) is 1839bp, and the cut lengths are 1083bp and 756bp respectively (the nucleotide sequences of the cut DNA fragments are SEQ ID NO.2 and SEQ ID NO.3 respectively).
(since there is little difference in the final results obtained by using D-cysteine and L-cysteine, only the results for D-cysteine are shown here).
Example 7: effect of pH on DNA cleavage results
100 μ L of the mixture obtained in example 4 was added to a cuvette with a stopper, the pH in the cuvette was adjusted to 6 or 8, respectively, and then the mixture was irradiated with left and right circular polarized lights obtained by adjusting quarter slides at different angles for 2 hours, respectively, using a 405nm laser as a light source. The irradiated mixture was centrifuged at 8000rpm for 10min and then characterized.
The characterization results are shown in FIG. 8, which shows that: after mixing the salmon DNA and the chiral semiconductor nano-particles, adjusting the pH value to be 2 or 8 respectively, irradiating with circularly polarized light for 2 hours respectively, and then breaking at the T and A basic groups in a GATATC specific fragment on the DNA sequence, wherein the original length of the salmon DNA (the nucleotide sequence is SEQ ID NO.1) is 1839bp, and then the cut lengths are 1083bp and 756bp respectively (the nucleotide sequences of the cut DNA fragments are SEQ ID NO.2 and SEQ ID NO.3 respectively).
(since there is little difference in the final results obtained by using D-cysteine and L-cysteine, only the results for D-cysteine are shown here).
Comparative example 1: incubation of chiral semiconductor nanoparticles with non-specific DNA
The sequence of the salmon DNA sequence at GATATC was changed to GATTAC to make it non-specific, and other parameters were kept consistent with examples 1-5 to see if the DNA sequence could still be cleaved.
As shown in FIG. 5, after mixing the nonspecific salmon DNA sequence with the chiral semiconductor nanoparticles and irradiating with circularly polarized light for 2 hours, no fragmentation of DNA occurred, indicating that no DNA cleavage occurred.
Comparative example 2: irradiation of semiconductor nanoparticles modified with different chiral ligands with DNA
During the synthesis process of the chiral semiconductor nanoparticles, the chiral ligand is replaced by glutathione from cysteine, and other parameters are consistent with those in examples 1-5, so that the chiral semiconductor nanoparticles modified by Glutathione (GSH) are obtained. The nanoparticles were then mixed with salmon DNA under the same incubation conditions and irradiated under polarized light for 2 hours under the same illumination conditions.
As a result, as shown in FIG. 6, the mixture of the glutathione-modified chiral semiconductor nanoparticles and salmon DNA was not subjected to DNA cleavage under light.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Sequence listing
<110> university of south of the Yangtze river
<120> method for shearing DNA molecule and application thereof
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 1839
<212> DNA
<213> Artificial sequence
<400> 1
atgcacccca ctacactcat cttaagctca tcccttttaa taatctttgc acttctaatc 60
tatcctctta tcaccactct cacccctacc cctcagcaca aaaactgatc ccttaaccaa 120
gtgaaaactg ccatcaaaat ggccttccta gtaagcttac tccccctttt tatcttccta 180
gatcaaggaa ctgaaactat cgtcactaac tgacaatgaa taaacaccac aacctttgat 240
attaacctta gctttaaatt tgaccactac tccattattt ttaccccgat cgccctgtac 300
gtaacctgat ctattctcga attcgcatca tggtacatac atgccgaccc caatataaac 360
cggttcttta aatatctcct cctcttcctg attgccataa ttattttagt caccgccaat 420
aacatatttc aactattcat cggctgagaa ggagttggaa ttatatcgtt cctcctcatt 480
gggtgatggc acggacgggc tgacgctaac acagctgcca tacaagctgt aatttataac 540
cgtgtaggag acatcggact tatcttgagt atggcctggt tcgcaataaa ccttaactcc 600
tgagaaattc aacaaatatt tgcctcttca aaaggactcg accttacact ccctcttatg 660
ggcctcattc tagccgccac cggcaaatca gcgcaatttg gacttcaccc gtgacttcct 720
tcagcgatag aaggtcctac gccggtatct gccctactac actccagcac catagtagtc 780
gcgggcatct tcctattaat tcgactccac cctcttatag aaaataacca aacagcccta 840
accacttgct tatgcctagg agccctaacc accctattca ccgctacctg tgccctaaca 900
caaaatgata ttaaaaaaat tgttgcattc tctacgtcca gtcaactagg acttatgata 960
gttaccatcg gacttaatca accacaacta gcctttctcc acatctgcac tcacgcattc 1020
ttcaaagcta tacttttctt atgctcgggc tcaattattc acagtttaaa cgacgaacaa 1080
gatatccgaa aaataggagg catacacaac ctcaccccat ttacttcctc ctgccttaca 1140
atcggaagcc ttgcacttac cggcaccccc ttcttagcag ggtttttctc taaagatgct 1200
attattgaag ccttaaacac ctcccacctc aacgcctggg ccctcactct taccttacta 1260
gccacctcat tcactgccat ttacagcctc cgagttatct ttttcgtctc tatgggacac 1320
ccccgcttta cgacaacggc ccctattaat gaaaataacc catccgtaat taacccaatc 1380
aagcggctag cctgggggag catcattgca ggactactaa ttacatcgaa tttcctccct 1440
accaacacac ccgtaataac tatgcccacc cacttgaaat tagccgctct cctggttacc 1500
atcttaggtc ttctcattgc attagaactt gcgtcactaa ctagcaagca atttaaaact 1560
acacccaaca ttatcacaca caacttctcc aacatgctag gattcttccc cgctatcatc 1620
caccgattaa ttcctaaact aaacttaact ctaggacaaa ccattgccag ccaaatggtt 1680
gatcaaacat gatttgaaaa agtcggcccg aaaggaatta tttcaacgca cctacccata 1740
gtcacaacga caagtaacat ccaacaaggc ataattaaaa catacctcac tctatttttc 1800
ctttcaacaa ctctagctgt tctactgaca ttaacctag 1839
<210> 2
<211> 1083
<212> DNA
<213> Artificial sequence
<400> 2
atgcacccca ctacactcat cttaagctca tcccttttaa taatctttgc acttctaatc 60
tatcctctta tcaccactct cacccctacc cctcagcaca aaaactgatc ccttaaccaa 120
gtgaaaactg ccatcaaaat ggccttccta gtaagcttac tccccctttt tatcttccta 180
gatcaaggaa ctgaaactat cgtcactaac tgacaatgaa taaacaccac aacctttgat 240
attaacctta gctttaaatt tgaccactac tccattattt ttaccccgat cgccctgtac 300
gtaacctgat ctattctcga attcgcatca tggtacatac atgccgaccc caatataaac 360
cggttcttta aatatctcct cctcttcctg attgccataa ttattttagt caccgccaat 420
aacatatttc aactattcat cggctgagaa ggagttggaa ttatatcgtt cctcctcatt 480
gggtgatggc acggacgggc tgacgctaac acagctgcca tacaagctgt aatttataac 540
cgtgtaggag acatcggact tatcttgagt atggcctggt tcgcaataaa ccttaactcc 600
tgagaaattc aacaaatatt tgcctcttca aaaggactcg accttacact ccctcttatg 660
ggcctcattc tagccgccac cggcaaatca gcgcaatttg gacttcaccc gtgacttcct 720
tcagcgatag aaggtcctac gccggtatct gccctactac actccagcac catagtagtc 780
gcgggcatct tcctattaat tcgactccac cctcttatag aaaataacca aacagcccta 840
accacttgct tatgcctagg agccctaacc accctattca ccgctacctg tgccctaaca 900
caaaatgata ttaaaaaaat tgttgcattc tctacgtcca gtcaactagg acttatgata 960
gttaccatcg gacttaatca accacaacta gcctttctcc acatctgcac tcacgcattc 1020
ttcaaagcta tacttttctt atgctcgggc tcaattattc acagtttaaa cgacgaacaa 1080
gat 1083
<210> 3
<211> 756
<212> DNA
<213> Artificial sequence
<400> 3
atccgaaaaa taggaggcat acacaacctc accccattta cttcctcctg ccttacaatc 60
ggaagccttg cacttaccgg cacccccttc ttagcagggt ttttctctaa agatgctatt 120
attgaagcct taaacacctc ccacctcaac gcctgggccc tcactcttac cttactagcc 180
acctcattca ctgccattta cagcctccga gttatctttt tcgtctctat gggacacccc 240
cgctttacga caacggcccc tattaatgaa aataacccat ccgtaattaa cccaatcaag 300
cggctagcct gggggagcat cattgcagga ctactaatta catcgaattt cctccctacc 360
aacacacccg taataactat gcccacccac ttgaaattag ccgctctcct ggttaccatc 420
ttaggtcttc tcattgcatt agaacttgcg tcactaacta gcaagcaatt taaaactaca 480
cccaacatta tcacacacaa cttctccaac atgctaggat tcttccccgc tatcatccac 540
cgattaattc ctaaactaaa cttaactcta ggacaaacca ttgccagcca aatggttgat 600
caaacatgat ttgaaaaagt cggcccgaaa ggaattattt caacgcacct acccatagtc 660
acaacgacaa gtaacatcca acaaggcata attaaaacat acctcactct atttttcctt 720
tcaacaactc tagctgttct actgacatta acctag 756
Claims (9)
1. A method for shearing DNA molecules is characterized in that chiral semiconductor nanoparticles and DNA molecules to be sheared are mixed and incubated, and then the incubated chiral semiconductor nanoparticles and DNA molecule mixture to be sheared are irradiated under circularly polarized light with the wavelength of 405nm to obtain sheared DNA molecules; the chiral semiconductor nano-particle is synthesized by taking D-type cysteine or L-type cysteine as a chiral ligand, and the DNA molecule to be sheared is a DNA molecule with a sequence containing GATATC specific segment; the method comprises the following specific steps:
(1) synthesis of a precursor: under the protection of nitrogen, reacting H2SO4To Al2Te3Obtaining precursor gas in the solution;
(2) adding Cd (ClO)4)2·6H2Adding O-type cysteine and D-type cysteine or L-type cysteine into water, introducing the precursor gas obtained in the step (1), stirring and heating the solution to obtain a nanoparticle solution, adding isopropanol into the obtained nanoparticle solution, performing centrifugal filtration to obtain a precipitate, and re-suspending the precipitate in PBS buffer solution to obtain chiral semiconductor nanoparticles;
(3) fully mixing the DNA molecules to be sheared with the chiral semiconductor nano particles obtained in the step (2) and then standing to obtain a mixed solution of the incubated chiral semiconductor nano particles and the DNA molecules;
(4) and (3) adding the mixed solution obtained in the step (3) into a cuvette with a plug, and irradiating the mixed solution by circularly polarized light by using 405nm laser as a light source to obtain the sheared DNA molecules.
2. The method for cleaving a DNA molecule according to claim 1, wherein in the step (3), the molar ratio of the DNA molecule to be cleaved to the chiral semiconductor nanoparticle is 45-55: 1.
3. The method of cleaving a DNA molecule according to claim 1 or 2, wherein the standing time in the step (3) is 0.5 to 1.5 hours.
4. The method of claim 1 or 2, wherein in step (4), the irradiation time is 1-3 h.
5. The method for cleaving a DNA molecule according to claim 1, wherein the standing time in the step (3) is 0.5 to 1.5 hours; in the step (4), the irradiation time is 1-3 h.
6. The method of claim 1, 2 or 5, wherein the circularly polarized light is left circularly polarized light and/or right circularly polarized light.
7. The method for cleaving a DNA molecule according to claim 1, wherein the standing time in step (3) is 0.5 to 1.5 hours, and the circularly polarized light is left circularly polarized light and/or right circularly polarized light.
8. The method of cleaving a DNA molecule according to claim 1, wherein the irradiation time in the step (4) is 1 to 3 hours, and the circularly polarized light is left circularly polarized light and/or right circularly polarized light.
9. Use of a method of cleaving a DNA molecule according to any one of claims 1 to 5 in genetic engineering.
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