CN104862302B - DNA fragmentation method and device for implementing same - Google Patents

DNA fragmentation method and device for implementing same Download PDF

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CN104862302B
CN104862302B CN201510223962.8A CN201510223962A CN104862302B CN 104862302 B CN104862302 B CN 104862302B CN 201510223962 A CN201510223962 A CN 201510223962A CN 104862302 B CN104862302 B CN 104862302B
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dna
sample
solution
fragmentation
bubbles
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CN104862302A (en
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水玲玲
彭亚运
李岚慧
金名亮
周国富
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South China Normal University
Shenzhen Guohua Optoelectronics Co Ltd
Academy of Shenzhen Guohua Optoelectronics
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Shenzhen Guohua Optoelectronics Co Ltd
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Abstract

The invention discloses a DNA fragmentation method and a device for realizing the method, wherein bubbles are continuously bubbled in a DNA solution, instantaneous fluid shearing force generated in the bubble breaking process acts on DNA molecules in the solution, and when the shearing force is greater than intermolecular force in a DNA molecular chain, the DNA molecules are sheared and broken, so that the fragmentation effect of the DNA molecules is achieved. The invention has the advantages of small volume of the required DNA sample, easy control of the experimental process, simple required device, low cost, simple operation, difficult blockage, no need of high pressure, no occurrence of a large amount of dead volume, no pollution to the sample, realization of random shearing of the DNA sample, relatively controllable fragment length, small fragment length dispersity and the like, and the sheared fragments of the DNA are uniformly distributed in a preset size.

Description

DNA fragmentation method and device for implementing same
Technical Field
The invention relates to a DNA fragmentation method and a device for realizing the method, in particular to a DNA fragmentation method which utilizes bubbles to shear DNA, has controllable length of DNA fragments and randomly breaks the DNA fragments.
Background
Randomly distributed DNA fragments are one of the key steps for techniques such as gene sequencing and DNA microarray-based analysis. For genetic analysis, the range of DNA fragment lengths is usually chosen to approximate the average length of a single gene in the organism of interest, or based on automated generation of sequence data for the entire database. In molecular diagnostics, different detection methods are used to screen part of a gene from a sample and to hybridize the target gene with a complementary probe molecule, so for molecular diagnostics applications DNA fragments are an important sample pre-treatment step, short DNA fragments are essential for rapid hybridization of DNA molecules and for highly sensitive target detection. With the progress of research, the demand for smaller, faster, more efficient, and less polluting real-time detection techniques is becoming greater.
Currently, the following methods are commonly used for DNA fragmentation: DNA restriction, sonication, spray atomization and hydrodynamic shearing. These methods are currently used for the generation of DNA fragments, but each method has its own advantages and disadvantages. The restriction endonuclease method has the advantages that the DNA can be cut at a specific position to obtain a fragment; but is susceptible to sample contamination, sample degradation, large length dispersion of the resulting DNA fragments, and causes relatively large amounts of single strand breaks. The sonication method, which generates a shear force using sound waves, can cause the breaking of long DNA strands, is fast and simple; but causes damage to the DNA sample, produces fragments with large length dispersion, and is difficult to integrate. The spray atomization method uses a pressurized nozzle to generate a mechanical shearing force to shear a DNA sample, and is fast and effective; however, the apparatus is expensive and requires a large amount of DNA sample, which is difficult to integrate into the apparatus. The method can generate fragment DNA based on the hydrodynamic force DNA fragment shearing method, the obtained DNA fragment has small length dispersion degree, the DNA of the double-chain fracture fragment can be obtained by the shearing of the method, the random fracture is realized, and the integration into the device is easy; however, the method has the disadvantages that the experimental system needs high pressure, the device is expensive to prepare, a large amount of dead volume exists, and the microstructure device is easy to block and is not suitable for miniaturization and integration.
It is of great practical interest to find a novel method for DNA fragmentation that overcomes some of the disadvantages of the prior art.
Disclosure of Invention
The invention aims to provide a DNA fragmentation method, which utilizes bubbles to shear DNA, the length of the DNA fragment is controllable, and the DNA fragment is randomly broken.
The technical scheme adopted by the invention is as follows:
a method of DNA fragmentation comprising the steps of:
1) preparing a sample: preparing a DNA original solution;
2) bubbling: inserting a gas outlet into the prepared DNA original solution, adjusting the air pressure, the size of bubbles and the bubble generation speed through an air pressure control device, continuously bubbling in the solution, and shearing long-chain DNA molecules into shorter fragments.
As a further improvement of the invention, the air pressure is adjusted to be 0.01-10Mpa by an air pressure control device.
As a further improvement of the invention, said conditioning bubbles willAdjusting the size of the bubbles to 10nm-10mm, and adjusting the generation speed of the bubbles to 1-10 per second6And (4) respectively. The smaller the generated bubble is, the larger the shearing force generated by the rupture of the bubble is, and therefore, the better the shearing effect is; the faster the bubble is generated, the faster it shears for the same amount of sample, the more efficient the shearing of DNA.
As a further improvement of the invention, the concentration of the DNA original solution is 1ug/mL-500 ug/mL.
As a further improvement of the invention, the bubbling time is 1-60 min.
A DNA fragmentation device for realizing the DNA fragmentation method comprises a gas supply device, a gas pressure control device, a gas outlet device and a connecting device, wherein the gas pressure control device is arranged at a gas outlet of the gas supply device, a gas outlet of the gas pressure control device is connected with a gas inlet of the gas outlet device through the connecting device, and a gas outlet of the gas outlet device is connected with the connecting device.
As a further improvement of the invention, the gas outlet device is a syringe or other airtight gas conducting device.
As a further improvement of the invention, the needle head of the syringe or the connector of the airtight gas conducting device is connected with a pipe with a corresponding size.
As a further improvement of the invention, the connecting device comprises a connecting air pipe with the same caliber as the air outlet of the air pressure control device and an air guide pipe with a certain diameter.
As a further improvement of the invention, the connecting device is a connecting device with good air tightness, and can ensure that gas can stably and continuously contact with the stock solution to continuously generate bubbles.
As a further improvement of the invention, the inner diameter of the air duct is 0.1-10 mm.
The invention has the beneficial effects that:
the invention provides a brand-new DNA fragmentation method, in particular to a DNA fragmentation method by shearing DNA by utilizing bubbles. The DNA sample required by the invention has small volume, the experimental process is easy to control, the required device is simple, the cost is low, the operation is simple, the blockage is not easy, the high pressure is not required, a large amount of dead volume can not appear, the sample pollution can not be caused, the random shearing of the DNA sample can be realized, the random shearing device has the advantages of relatively controllable fragment length, small fragment length dispersity and the like, the DNA sheared fragments are uniformly distributed in the preset size, and the DNA fragments in the size range of 100bp-50kbp can be sheared under different pressures. The DNA fragmentation method can realize cheap, rapid and effective random DNA fragmentation, and the obtained DNA fragment can be applied to the technologies of DNA molecule rapid hybridization, high-sensitivity molecule diagnosis, genome sequencing and the like.
Drawings
FIG. 1 is a schematic diagram of an apparatus for DNA fragmentation.
FIG. 2 is a photograph of gel electrophoresis before and after DNA fragmentation. (from right to left are DNA marker, intact DNA sample and DNA sample after bubble fragmentation treatment, respectively.)
FIG. 3 is a photograph of gel electrophoresis after DNA fragmentation of salmon sperm. (from right to left are DNA marker, intact DNA sample and DNA sample after bubble fragmentation treatment, respectively.)
FIG. 4 is a photograph of gel electrophoresis of herring sperm after DNA fragmentation. (from right to left are DNA marker, intact DNA sample and DNA sample after bubble fragmentation treatment, respectively.)
Description of reference numerals:
1-a gas supply device; 2-a pneumatic control device; 3-an air outlet device; 4-vial; 5-DNA stock solution; 6-connecting device.
Detailed Description
The technical solution of the present invention will be further described with reference to the following embodiments.
It should be noted that the embodiments are only used for illustrating the technical solutions of the present invention, and should not be considered as limiting the scope of the present invention.
Example 1
Referring to fig. 1, a DNA fragmentation device comprises a gas supply device 1, a gas pressure control device 2, a gas outlet device 3 and a connecting device 6, wherein the gas pressure control device 2 is arranged at a gas outlet of the gas supply device 1, the gas outlet of the gas pressure control device 2 is connected with a gas inlet of the gas outlet device 3 through the connecting device 6, and the gas outlet of the gas outlet device 3 is connected with the connecting device 6 and inserted into a DNA raw solution 5 in a vial 4 through the connecting device 6. The gas supply device 1 is a gas storage tank or a gas transmission pump, and the gas outlet device 3 is an injector or a closed gas conduction device. The connector of the air outlet device 3, namely the connector of the needle head of the injector or the connector of the airtight gas conduction device, is connected with a pipe with a corresponding size. The connecting device 6 comprises a connecting air pipe with the same caliber as the air outlet of the air pressure control device 2 and an air guide pipe with a certain diameter, and the inner diameter of the air guide pipe is 0.1-10 mm. The connecting device 6 has good air tightness, and ensures that gas can stably and continuously contact with stock solution to continuously generate bubbles.
DNA fragmentation was performed using the DNA fragmentation apparatus described above:
1) generating bubbles: selecting a fixed gas supply device 1, and starting the gas supply device 1;
2) preparing a sample: preparing a certain amount of DNA original solution with a certain concentration, wherein the method has no special requirements on the type, concentration and the like of a sample, the DNA of various long fragments including genome DNA solution is suitable, the concentration of the DNA original solution is 1ug/mL-500ug/mL, and when the concentration of the DNA original solution is 100 ug/mL, the volume of the DNA solution is not less than 0.01 mL;
3) bubbling: inserting an air blowing outlet into the DNA original solution 5 sample, adjusting air pressure to 0.01-10Mpa by adjusting the air pressure control device 2, adjusting the size of air bubbles to 10nm-10mm, and generating air bubbles at a speed of 1-10 per second6Continuously generating bubbles, breaking the generated bubbles or rising the bubbles to the surface of the liquid for breaking, generating instantaneous fluid shear force in the process of breaking the bubbles, acting on the DNA molecules in the solution, when the shear force is larger than the intermolecular force in the DNA molecular chains, DThe NA molecules are cut and broken to form short DNA molecule fragments, so that the fragmentation effect of the DNA molecules is achieved, and when the generated bubbles are smaller, the shearing force generated by the breakage of the bubbles is larger, and the shearing effect is better; the larger the speed of generating bubbles is, the faster the shearing speed of the sample with the same quantity is, the higher the efficiency of shearing DNA is, and the bubbling time is controlled to be 1-60 min;
4) and (3) detecting the length of the DNA fragment: the general DNA fragment detection method is applicable, and the agarose gel electrophoresis analysis method is taken as an example here, and the length detection and analysis are carried out on the sheared DNA fragment: the sample was added to the prepared agarose gel, electrophoresis was performed with an electrophoresis apparatus, and after electrophoresis, DNA bands were observed with a SIM gel analyzer and software and photographed to obtain experimental results as shown in fig. 2.
Example 2
Salmon sperm DNA fragmentation: DNA fragmentation was performed by the DNA fragmentation method described in example 1 using salmon sperm DNA solution, which was a commercially available sample of CAS: 68938-01-2, the DNA concentration of the sample is 10-500ug/mL, the volume of the selected solution is 0.1-100mL, the gas pressure is 0.01-10Mpa, the tube diameter of the gas-guide tube is 0.1-10mm, and the bubble shearing time is 1-60 min. The cut DNA fragments were subjected to length detection and analysis, and the results are shown in FIG. 3. As can be seen from FIG. 3, salmon sperm DNA has been cleaved into a DNA fragment of 500 bp to 7500 bp.
Example 3
Herring sperm DNA fragmentation: the herring sperm DNA solution was used to fragment DNA according to the DNA fragmentation method described in example 1, the DNA solution selected was a commercially available sample with product number CAS: 9007-49-2, the DNA concentration of the sample is 1-500ug/mL, the volume of the selected solution is 1-100mL, the gas pressure is 0.01-10Mpa, the tube diameter of the used gas-guide tube is controlled to be 0.1-10mm, and the bubble shearing time is 1-60 min. The cut DNA fragments were subjected to length detection and analysis, and the results are shown in FIG. 4. As can be seen from FIG. 4, herring sperm DNA has been spliced into DNA fragments of 500 bp to 5000 bp.

Claims (3)

1. A method of DNA fragmentation comprising the steps of:
1) preparing a sample: preparing a DNA original solution;
2) bubbling: inserting the gas outlet into the prepared DNA original solution, regulating the air pressure to 0.01-10Mpa and the size of the bubbles to 10nm-10mm by an air pressure control device, and regulating the bubble generation speed to 1-10 per second6Continuously bubbling through the solution, shearing long-chain DNA molecules into shorter fragments.
2. The method of claim 1, wherein the concentration of the DNA stock solution is 1ug/mL-500 ug/mL.
3. The method of claim 1, wherein the bubbling time is 1-60 min.
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