CN108998442B - A kind of method for shearing DNA molecule and its application - Google Patents

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

A kind of method for shearing DNA molecule and its application

technical field

The invention relates to a method for shearing DNA molecules and its application, and belongs to the technical field of genetic engineering.

Background technique

Obtaining DNA fragments is a critical step for technologies such as gene sequencing, gene analysis, and gene editing. For gene sequencing, it can efficiently obtain target fragments; for gene analysis, it can obtain single gene information; for gene editing, it can selectively extract or copy gene sequences. Therefore, the shearing of DNA molecules is an essential preprocessing step in the field of genetic engineering.

With the deepening of research in the field of genes, the demand for obtaining DNA fragments with uniform structure and high specificity in sequence and site has become more and more important.

At present, the commonly used methods for shearing DNA molecules are as follows: DNA restriction enzyme digestion, ultrasonic degradation, hydrodynamic shearing, etc. These methods are currently used for the generation of DNA fragments, but the restriction The cleavage method still requires specific biological enzymes and has the defects of high pH and temperature requirements; the ultrasonic degradation method and the hydrodynamic shearing method have the defect of being non-specific to the target sequence.

Therefore, none of the above methods can well obtain DNA fragments with uniform structure and high specificity in sequence and site. We urgently need to find a DNA fragment that can obtain uniform structure and high specificity in sequence and site. method to meet the needs of research in the field of genetics.

SUMMARY OF THE INVENTION

Chiral semiconductor nanoparticles are composed of IV, II-VI, IV-VI or III-V elements, and are often used in sensing, imaging, catalysis, etc.; circularly polarized light is the direction of the electric field of light or the end of the light vector that propagates The trajectory drawn on the plane of the direction is often used for the characterization of chiral active substances.

To solve the above problems, the present invention provides a method for shearing DNA molecules. This method combines chiral semiconductor nanoparticles with circularly polarized light. Using the special configuration of chiral nanoparticles and the principle that they are prone to photo-oxidation under light, the chiral semiconductor nanoparticles are first mixed with DNA molecules and then incubated. , and then irradiate the incubated mixture of chiral semiconductor nanoparticles and DNA molecules under circularly polarized light to obtain sheared DNA molecules; using this method, a large number of uniform structures can be quickly obtained, and the DNA fragments with high specificity have great application potential in the field of genetic engineering.

The technical scheme of the present invention is as follows:

The invention provides a method for shearing DNA molecules. The method comprises the steps of first mixing chiral semiconductor nanoparticles and DNA molecules and then incubating them, and then exposing the incubated mixture of chiral semiconductor nanoparticles and DNA molecules to circularly polarized light. Under irradiation, the sheared DNA molecule is obtained.

In one embodiment of the present invention, the method includes dissolving DNA molecules in a PBS buffer to obtain a DNA molecule solution; adding chiral semiconductor nanoparticles into the DNA molecule solution for mixing to obtain a mixed solution; Let stand for incubation to obtain an incubated mixed solution; irradiate the incubated mixed solution under circularly polarized light to obtain sheared DNA molecules.

In an embodiment of the present invention, the concentration of the PBS buffer is 0.01 mol/L.

In one embodiment of the present invention, the pH of the PBS buffer is 7.4.

In an embodiment of the present invention, the concentration of DNA molecules in the DNA molecule solution is 0.5-1.5 μmol/L.

In an embodiment of the present invention, the concentration of DNA molecules in the DNA molecule solution is 1 μmol/L.

In an embodiment of the present invention, the chiral semiconductor nanoparticles are synthesized by using D-cysteine or L-cysteine as a chiral ligand.

In an embodiment of the present invention, the molar ratio of the chiral semiconductor nanoparticles to DNA molecules is 45-55:1.

In an embodiment of the present invention, the molar ratio of the chiral semiconductor nanoparticles to DNA molecules is 50:1.

In an embodiment of the present invention, the standing time is 0.5-1.5 h.

In an embodiment of the present invention, the standing time is 1 h.

In an embodiment of the present invention, the irradiation is to add the incubated mixture into a cuvette with a stopper first, and then irradiate under circularly polarized light.

In an embodiment of the present invention, the irradiation time is 1-3 hours.

In an embodiment of the present invention, the irradiation time is 2 hours.

In an embodiment of the present invention, the circularly polarized light is left circularly polarized light and/or right circularly polarized light.

In an embodiment of the present invention, the light source of the circularly polarized light is a laser.

In an embodiment of the present invention, the light source of the circularly polarized light is a laser with a wavelength of 405 nm.

In one embodiment of the invention, the circularly polarized light is obtained by passing the light source through quarter glass slides at different angles.

The present invention provides a DNA molecule fragment obtained by shearing the above-mentioned method for shearing a DNA molecule.

The present invention provides the application of the above-mentioned method for shearing DNA molecules in genetic engineering.

Beneficial effects:

(1) Using the method of the present invention, a large number of DNA fragments with uniform structure and high specificity in sequence (GAT-ATC) and site (between T-A) can be quickly obtained, which has great application potential in the field of genetic engineering ;

(2) The chiral nanoparticles used in the present invention are insensitive to temperature and pH, and can be applied to DNA shearing under various complex conditions;

(3) The chiral nanoparticles used in the present invention have high specificity and biocompatibility, and can generate DNA-specific cleavage in living cells and tumors, and have potential applications in clinical disease treatment.

Description of drawings

Fig. 1 TEM image of chiral semiconductor nanoparticles;

Fig. 2 circular dichroism spectrum of chiral semiconductor nanoparticles;

Fig. 3 Electropherograms of the mixture of Salmon DNA and L-Cys CdTe under right circularly polarized light irradiation for 2 h;

Fig. 4 Electropherogram of the mixture of Salmon DNA and D-Cys CdTe under left circularly polarized light before and after irradiation for 2 h;

Fig. 5 electrophoresis images of the mixture of non-specific Salmon DNA sequence and L-Cys CdTe under right circularly polarized light irradiation for 2 h;

Fig. 6 Electropherograms of the mixture of Salmon DNA and L-GSH CdTe under circularly polarized light irradiation for 2 h;

Figure 7 Electropherograms of the mixture of Salmon DNA and L-GSH CdTe at (a) 0 degrees Celsius (b) 50 degrees Celsius and irradiated under circularly polarized light for 2 hours before and after;

Figure 8 Electropherograms of a mixture of Salmon DNA and L-GSH CdTe in (a) pH 6 (b) pH 8 solution and irradiated under circularly polarized light for 2 h before and after.

Detailed ways

Taking Salmon DNA as an example below, the present invention will be further elaborated in conjunction with specific examples and comparative examples.

The characterization method involved in the present invention is as follows:

Characterization method of DNA shearing effect: Take 20 uL of sheared DNA product and mix it with loading buffer (InvitrogenTM). It was then quickly added to a pre-prepared 2.5% agarose gel only. After the sample settled to the bottom of the gel hole, the gel was put into electrophoresis buffer (Invitrogen ^™ ) and run at 110V for 30min. Finally, DNA bands were detected on a gel imager.

Electron microscope characterization method: drop the liquid sample on the carbon support membrane, and remove the sample with absorbent paper after 5 min. The copper mesh was then placed in the TEM sample holder, and the sample was observed at an accelerating voltage of 220KV.

Example 1: Synthesis of Precursors

Under the protection of nitrogen, 4 mL of 0.5 M H ₂ SO ₄ was added to 0.05 g of Al ₂ Te ₃ solution to obtain the precursor gas.

Example 2: Synthesis of Chiral Semiconductor Nanoparticles

After adding 0.985g of Cd(ClO ₄ ) ₂ .6H ₂ O and 3mL of 1M D-cysteine to 125mL of water, the precursor gas obtained in Example 1 was introduced, and NaOH was used to adjust the pH to 12. The precursor gas was Nitrogen was used as the carrier gas, and the flow rate was controlled at 100 mL/min. The solution was heated to 110 °C while stirring and kept for 8 h. Then, isopropanol was added to the obtained nanoparticle solution in a volume ratio of 1:1, and then the mixture was mixed. Centrifuge at 10,000 rpm for 5 min. The pellet was then resuspended in PBS buffer (0.01 M, pH 7.4) 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 diagram of chiral semiconductor nanoparticles)

Example 3: Synthesis of Chiral Semiconductor Nanoparticles

After adding 0.985g of Cd(ClO ₄ ) ₂ .6H ₂ O and 3mL of 1M L-cysteine to 125mL of water, the precursor gas obtained in Example 1 was introduced, and the pH was adjusted to 12 with NaOH. Nitrogen was used as the carrier gas, and the flow rate was controlled at 100 mL/min. The solution was heated to 110 °C while stirring and kept for 8 h. Then, isopropanol was added to the obtained nanoparticle solution in a volume ratio of 1:1, and then the mixture was mixed. Centrifuge at 10,000 rpm for 5 min. The pellet was then resuspended in PBS buffer (0.01 M, pH 7.4) 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 diagram of chiral semiconductor nanoparticles)

Example 4: Incubation of Chiral Semiconductor Nanoparticles with DNA

50 μL of salmon sperm DNA (Salmon DNA) solution with nucleotide sequence of SEQ ID NO. The chiral semiconductor nanoparticles obtained in Examples 2 and 3 were thoroughly mixed in a molar ratio of 50:1 and then allowed to stand for 1 hour to obtain a mixed solution of the incubated chiral semiconductor nanoparticles and DNA.

Example 5: Irradiation of chiral semiconductor nanoparticles and DNA

Take 100 μL of the mixture obtained in Example 4 and add it to a stoppered cuvette, then use a 405nm laser as a light source, and irradiate the mixture with left and right circularly polarized lights obtained by adjusting quarter glass slides at different angles. 2h.

The irradiated mixture was centrifuged at 8000 rpm for 10 min for characterization.

The characterization results are shown in Figure 3-4. It can be seen from the figure that after mixing salmon DNA with chiral semiconductor nanoparticles and irradiating with circularly polarized light for 2 hours, a break occurs at the T and A bases in the GATATC-specific fragment on the DNA sequence. , salmonDNA (nucleotide sequence is SEQ ID NO.1) original length is 1839bp and the length after shearing is 1083bp and 756bp respectively (the nucleotide sequence of DNA fragment after shearing is SEQ ID NO.2, SEQ ID NO. .3).

(Because there is almost no difference between the final results obtained by using D-cysteine and L-cysteine, only the results of D-cysteine are used here)

Example 6: The effect of temperature on DNA shearing results

Take 100 μL of the mixture obtained in Example 4 and add it to a stoppered cuvette, and place the cuvette in an environment of 0 degrees and 50 degrees respectively, and then use a 405nm laser as the light source, and adjust the quarters of the different angles. The left and right circularly polarized light obtained from a glass slide were respectively irradiated to the mixture for 2 h. The irradiated mixture was centrifuged at 8000 rpm for 10 min for characterization.

The characterization results are shown in Figure 7. It can be seen from the figure that after mixing salmon DNA with chiral semiconductor nanoparticles and irradiating circularly polarized light at 0 degrees and 50 degrees for 2 hours, the T in the GATATC-specific fragment on the DNA sequence A break occurs at base A, and the original length of salmon DNA (nucleotide sequence is SEQ ID NO. 1) is 1839bp and the length after shearing is 1083bp and 756bp respectively (the nucleotide sequence of the DNA fragment after shearing is SEQ ID NO.1, respectively. ID NO.2, SEQ ID NO.3).

(Since the final results obtained by using D-cysteine and L-cysteine are almost indistinguishable, only the results of D-cysteine are used here).

Example 7: The effect of pH on DNA shearing results

Take 100 μL of the mixture obtained in Example 4 and add it to a stoppered cuvette, adjust the pH in the cuvette to 6 or 8, and then use a 405nm laser as the light source to adjust the quarter glass slides at different angles. The left and right circularly polarized light were respectively irradiated to the mixture for 2 h. The irradiated mixture was centrifuged at 8000 rpm for 10 min for characterization.

The characterization results are shown in Figure 8. It can be seen from the figure that after mixing salmon DNA with chiral semiconductor nanoparticles and adjusting the pH to 2 or 8, respectively, after irradiating with circularly polarized light for 2 hours, T and A break occurs at base A, and the original length of salmon DNA (nucleotide sequence is SEQ ID NO. 1) is 1839 bp, and the length after shearing is 1083 bp and 756 bp respectively (the nucleotide sequence of the sheared DNA fragment is SEQ ID NO. 1). NO.2, SEQ ID NO.3).

(Since the final results obtained by using D-cysteine and L-cysteine are almost indistinguishable, only the results of D-cysteine are used here).

Comparative Example 1: Incubation of Chiral Semiconductor Nanoparticles with Nonspecific DNA

The sequence of the salmon DNA sequence at GATATC was changed to GATTAC to make it a non-specific sequence, and other parameters were kept the same as those in Example 1-5, and it was observed whether the DNA sequence could still be cut.

The results are shown in Figure 5. After the non-specific salmon DNA sequence was mixed with the chiral semiconductor nanoparticles and irradiated with circularly polarized light for 2 hours, no fragmentation of the DNA occurred, indicating that no DNA shearing occurred.

Comparative example 2: Irradiation of semiconductor nanoparticles modified with different chiral ligands and DNA

During the synthesis of chiral semiconductor nanoparticles, the chiral ligand was replaced by cysteine with glutathione. semiconductor nanoparticles. The nanoparticles were then mixed with salmon DNA under the same incubation conditions and irradiated with polarized light for 2 hours under the same lighting conditions.

The results are shown in Figure 6. The mixture of glutathione-modified chiral semiconductor nanoparticles and salmon DNA did not undergo DNA cleavage under illumination.

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone who is familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, The protection scope of the present invention should be defined by the claims.

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Claims (9)

1. a method for shearing DNA molecule, it is characterized in that, described method is to hatch after mixing chiral semiconductor nanoparticle and DNA molecule to be sheared first, then hatch the chiral semiconductor nanoparticle to be sheared. The cut DNA molecule mixture is irradiated under circularly polarized light with a wavelength of 405 nm to obtain the cut DNA molecule; the chiral semiconductor nanoparticles are chiral with D-cysteine or L-cysteine Ligand synthesis, the DNA molecule to be sheared is a DNA molecule containing GATATC-specific fragments in sequence; the specific steps are as follows: (1) Synthesis of precursor: under the protection of nitrogen, H ₂ SO ₄ was added to the Al ₂ Te ₃ solution to obtain the precursor gas; (2) After adding Cd(ClO ₄ ) ₂ ·6H ₂ O and D-cysteine or L-cysteine into water, pass the precursor gas obtained in step (1), and heat the solution while stirring Obtaining a nanoparticle solution, adding isopropanol to the obtained nanoparticle solution, centrifugal filtration to obtain a precipitate, and resuspending the precipitate in a PBS buffer to obtain chiral semiconductor nanoparticles; (3) fully mixing the DNA molecules to be sheared and the chiral semiconductor nanoparticles obtained in step (2), and then standing to obtain a mixed solution of the incubated chiral semiconductor nanoparticles and DNA molecules; (4) adding the mixed solution obtained in step (3) into a cuvette with a stopper, using a 405 nm laser as a light source, and irradiating the mixed solution with circularly polarized light to obtain sheared DNA molecules. 2. The method for shearing DNA molecules according to claim 1, wherein in step (3), the molar ratio of the DNA molecules to be sheared to the chiral semiconductor nanoparticles is 45- 55:1. 3 . The method for shearing DNA molecules according to claim 1 or 2 , wherein, in step (3), the standing time is 0.5-1.5 h. 4 . 4 . The method for shearing DNA molecules according to claim 1 or 2 , wherein, in step (4), the irradiation time is 1-3 h. 5 . 5 . The method for shearing DNA molecules according to claim 1 , wherein in step (3), the standing time is 0.5-1.5 h; in step (4), the irradiated The time is 1-3h. 6. The method for shearing DNA molecules according to 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 shearing DNA molecules according to claim 1 , wherein, in step (3), the standing time is 0.5-1.5 h, and the circularly polarized light is Left circularly polarized light and/or right circularly polarized light. 8 . The method for shearing DNA molecules according to claim 1 , wherein, in step (4), the irradiation time is 1-3 hours, and the circularly polarized light is a left circular light. 9 . Polarized light and/or right circularly polarized light. 9. Application of the method for shearing DNA molecules according to any one of claims 1-5 in genetic engineering.

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