CN112251431A - Sample processing method, extraction kit and extraction device for rapidly extracting biological DNA - Google Patents

Abstract

The invention provides a biological sample processing method for rapidly extracting biological tissue DNA, which comprises the following steps: after simple treatment of the sample, grinding by a grinder, transferring the treated biological tissue sample into DNA lysate, adding isopropanol to form nano DNA, transferring into a DNA micro-column, cleaning and eluting. The biological sample treatment method provided by the invention is used for quickly extracting the DNA of the biological tissue, can greatly shorten the operation time, simplifies the operation steps, improves the experiment efficiency, does not use toxic reagents, is beneficial to the health of operators and reduces the damage to the environment.

Description

Sample processing method, extraction kit and extraction device for rapidly extracting biological DNA

[ technical field ] A method for producing a semiconductor device

The invention belongs to the technical field of biological engineering, and particularly relates to a processing method for quickly extracting a sample from biological tissue DNA, an extraction kit and an extraction device.

[ background of the invention ]

With the development of genome sequencing technology, the sequence, structure and function research of biological genome is rapidly developed, and obtaining biological genome DNA with high purity, high content and high integrity is the first prerequisite for the application of genome sequencing technology.

The traditional biological genome DNA extraction method mainly comprises a CTAB method and an SDS method, and the extraction steps mainly comprise grinding biological tissues under the protection of liquid nitrogen or treating the biological tissues with Protein K, and then carrying out processes of cell lysis, impurity removal, DNA precipitation, rinsing, elution and the like. It mainly uses detergents such as CTAB or SDS to crack cells and releases genome DNA in the cells; then removing impurities such as protein, polyphenol, polysaccharide and the like by a method of chloroform and phenol extraction or high-salt precipitation; then adding a proper volume of organic solvent such as absolute ethyl alcohol, isopropanol or PEG and the like into the extracted supernatant to precipitate or adsorb the DNA on media such as a silica gel column, an ion exchange column and the like; finally, after rinsing with a rinsing solution, the solution is dissolved by a low-concentration salt solution or eluted from the medium.

Patent document CN 110592072a discloses a method for extracting plant genome DNA and its application, which comprises grinding biological tissue under the protection of liquid nitrogen, adding biological tissue powder into CTAB lysate at 65 ℃ and incubating for 40 minutes, then extracting DNA, the whole operation time is more than 2.5 hours, and toxic substances such as beta-mercaptoethanol are needed.

Patent document CN 110358762a discloses a method for extracting plant leaf genome DNA, which comprises placing plant leaves into a multichannel homogenizer for disruption, incubating for 45 minutes at 65 ℃ in a lysis solution, and extracting DNA by a magnetic bead method, wherein the total operation time is not less than 1 hour, toxic substances such as beta-mercaptoethanol and guanidine hydrochloride are required, and the requirements for instruments are complex.

Patent document CN 110592072a discloses a method for extracting plant genomic DNA and its application, which comprises grinding plant tissue under the protection of liquid nitrogen, adding plant tissue powder into lysis solution containing SDS, and incubating at 65 ℃ for 30-50 minutes. And (2) placing the DNA for 5-10 minutes at the temperature of-20 ℃, then extracting the DNA, wherein the total operation time is about 2 hours, and toxic substances of beta-mercaptoethanol are required.

Patent document CN 106754889 a discloses a method for extracting high quality genomic DNA from a Chinese medicinal plant tissue, which comprises repeatedly extracting plant tissue cell nuclei at low temperature. Then, proteinase K and SDS lysates were added and digested overnight at 37 ℃ which took too long. And phenol and chloroform toxic substances are used.

Patent document CN 104694530 a discloses a method for extracting high quality genomic DNA from wheat leaf tissue by using a 96-well plate grinder, which comprises low temperature sample treatment, grinding, and then completing the DNA extraction process by using an extraction reagent. But the sample handling process is too time consuming: 20-28 h, the extraction process is also very time-consuming, more than 2h, and toxic substances are also used, such as: chloroform, phenol, and the like.

As can be seen from the above patent documents, the existing methods for extracting DNA from biological tissues generally have the problems of long operation time (generally longer than 1 hour or even 2 hours), use of toxic substances (phenol, chloroform, β -mercaptoethanol), and the like, and the long operation time causes degradation of DNA, wastes precious time of experimenters, and reduces experimental efficiency; the use of toxic substances can cause harm to physical and mental health of operators, the waste discharge has adverse effect on the environment, the post-treatment cost is high, and the requirements of sustainable development are not met.

[ summary of the invention ]

The present invention is directed to overcoming the above-mentioned deficiencies of the prior art and providing a sample processing method capable of rapidly extracting DNA from a biological tissue, and a kit and an extraction apparatus for performing the method.

In order to achieve the above object, the present invention provides a sample processing method for rapidly extracting DNA from a biological tissue, comprising the steps of:

(a) adding the simply treated biological tissue into a grinding device or a grinding instrument;

(b) sufficiently grinding the biological tissue;

(c) transferring the ground sample into a DNA extraction solution for DNA extraction.

The organisms include several plant tissues selected at random: leaf of Cymbopogon variegates, leaf of Poplar, leaf of willow and leaf of China rose; several animal tissues: liver of snakehead, liver of mouse and lung of mouse; a microorganism which: a yeast.

The biological DNA extraction reagent randomly selects three kits: triton X-100 DNA kit, Tujel DNA kit and Vickers DNA kit.

The grinding device is selected from biological tissue disruption devices commonly used in the art, typically a MY-10 grinder and a MY-20 grinder are preferred, but other devices capable of achieving the same purpose may be employed.

As a preferred option, the biological sample described in step (a) requires simple pre-processing, such as: cutting the plant leaves into smaller leaf tissues.

As a preferred embodiment, the plant tissue and yeast are treated with a MY-10 grinder in step (b); animal tissue was treated with a MY-20 abrader. Grinding can be carried out under the protection of a cracking solution, under the protection of liquid nitrogen, or by combining the two methods.

As a preferred embodiment, step (c) further comprises:

i. transferring the treated biological tissue into a container (typically an EP tube) containing a first Triton X-100 DNA lysate (Fuile DNA lysate or Vickers DNA lysate) containing RNase A, uniformly mixing (preferably, uniformly mixing for 10-30 seconds by vortex shaking), and then placing for 2-8 minutes, such as the Triton X-100 DNA lysate and the Fuile DNA lysate; adding the second solution if necessary, uniformly mixing, standing at room temperature for 2-8 minutes, and adding the second solution if the ground biological sample tissue is transferred into Vickers DNA lysate to be uniformly mixed;

centrifuging (preferably at 12000rpm, about 13400g for 1-2 minutes) and retaining the supernatant (preferably transferring the supernatant to a new centrifuge tube);

adding a second solution (such as a Vickers DNA kit needs to be added with a third solution) and uniformly mixing, transferring into an adsorption column, centrifuging (preferably, at the rotation speed of 12000rpm, about 13400g, and centrifuging for 20-40 seconds), and discarding the filtrate;

adding a third solution (such as a fourth solution is required to be added in a Vickers DNA kit) to the adsorption column, centrifuging (preferably, rotating at 12000rpm, about 13400g, centrifuging for 20-40 seconds), and discarding the filtrate;

v. fully drying the adsorption column (preferably, standing for 1-3 minutes at room temperature), and transferring to a new container;

and vi, dropwise adding a fourth solution (such as the fifth solution is required to be added in the Vickers DNA kit) to the center of the adsorption column, standing at room temperature (preferably, standing for 1-3 minutes), centrifuging (preferably, at a rotating speed of 12000rpm for about 13400g for 30 seconds-2 minutes), and collecting filtrate to obtain the biological tissue DNA.

The adsorption column is selected from DNA adsorption columns commonly used in nucleic acid extraction in the field, typically a silica membrane adsorption column, which has strong adsorption effect on DNA under acidic conditions, and has significantly reduced adsorption effect on DNA under alkaline conditions, and is commonly used in DNA extraction.

As a preferred scheme, the steps i to vi are all completed in a room temperature environment; to increase DNA purity, step iv can be repeated several times, typically 1, 2 or 3 times; in order to increase the DNA yield, step vi can be repeated 1 time, i.e., the filtrate is added to the same adsorption column again and centrifuged.

Preferably, the first solution is a lysate, a Triton X-100 DNA lysate (a fuelile DNA lysate or a vkele DNA lysate) containing RNase a, and the second solution of the vkele DNA kit is 5M NaCl.

Preferably, the second solution is isopropanol (a third solution of a Vickers DNA kit), and the ratio of the addition amount of the isopropanol to the volume of the supernatant obtained in the step ii is 0.6-1.0: 1; the third solution is rinsing liquid (fourth solution of the Vickers DNA kit) which contains 65-80% of ethanol; the fourth solution is an eluent TE (fifth solution of the Vickers DNA kit), the pH value of the fourth solution is in the range of 7.0-8.5, and if the pH value of deionized water obtained by a water making machine is in the range, the fourth solution can be directly eluted by pure water.

Through the above operation, the extraction of the biological tissue DNA can be completed within 20 minutes.

The carrier for describing the method for processing a sample of biological tissue DNA is typically a paper specification, and may be any carrier for describing an electronic version of the method (including but not limited to a removable magnetic disk, an optical disk, an electronic ink screen, a network resource, an address thereof, and the like), as long as the method can be known by reading the carrier.

In another aspect, the present invention provides a computer-readable medium carrying a computer program for implementing the method for processing a sample of biological tissue DNA. The computer is understood in a broad sense and includes but is not limited to a single chip microcomputer, a PLC, a single chip microcomputer, an industrial personal computer, a PC and the like. The computer readable carrier includes, but is not limited to, any form of Flash, EEPROM, magnetic disk (floppy or hard disk), optical disk, and the like. The computer program may be written in any language, such as assembly, JAVA, VB, VC, C + +, Python, as long as the associated system is controlled to implement the method.

The inventor surprisingly found in the research of the sample processing method for extracting the DNA of the biological tissue that after the biological tissue is processed by the MY-10 grinder or the MY-20 grinder, the particles of the tissue sample reach a very small degree, and the DAN of the biological tissue rapidly enters the extracting solution in a short time, so that the aim of rapidly extracting the DNA is fulfilled.

The invention provides a processing method, an extraction kit and an extraction device for extracting a biological tissue DNA sample, which can at least produce the following beneficial effects: through the treatment of the grinding instrument, the biological sample particles become small, the surface area becomes large, DNA can more easily enter the DAN extracting solution, the operation time is greatly shortened, the operation steps are simplified, and the experiment efficiency is improved; toxic reagents are not used, so that the method is green and environment-friendly and is beneficial to the health of researchers; most of the experimental work was done at room temperature.

[ description of the drawings ]

FIG. 1 is a gel electrophoresis image of four different plant DNA extracts from three different DNA kits;

FIG. 2 is a gel electrophoresis image of three different DNA kits for extracting DNA from three animal tissues and yeast;

[ detailed description ] embodiments

The invention is further described below in conjunction with the drawings and the specific embodiments, which are provided only to assist in understanding the invention.

Example 1 Rapid extraction of four plant DNAs from three DNA cassettes

As described above, 150ul Triton X-100 DNA lysate (either scribble Happy DNA lysate or Vickers DNA lysate) was placed in a 1.5ml EP tube, and cut young plant tissue was added and ground thoroughly using a MY-10 grinder. Then, 450ul Triton X-100 DNA lysate (Fule DNA lysate or 350ul Vickers DNA lysate) was added and mixed well. Or putting tender plant tissue into an EP tube, adding a small amount of liquid nitrogen into the EP tube, and fully grinding by using a MY-10 grinder under the protection of the liquid nitrogen outside the EP tube. Plant DNA was extracted according to the method described previously, and the results were determined on a biaosharp (Table 1, Table 2 and Table 3) and detected by gel electrophoresis (FIG. 1). As can be seen, the sample treated by the MY-10 grinding instrument can realize the rapid extraction of the plant genome DNA.

Table 1: quality of DNA extracted from plant tissue by scribble happiness DNA kit

Name of plant	Leaf of Maohua Bauhinian	Willow leaf	Poplar leaf	Chinese rose leaf
					Plant tissue quality	98.5mg	99.7mg	105.3mg	101.8mg
DNA concentration	152.4ng/ul	131.6ng/ul	187.2ng/ul	173.1ng/ul
					A260/A280	1.864	1.857	1.863	1.872

Table 2: quality of plant tissue DNA extracted by Vickers DNA kit

Name of plant	Leaf of Maohua Bauhinian	Willow leaf	Poplar leaf	Chinese rose leaf
					Plant tissue quality	100.9mg	104.1mg	102.9mg	96.1mg
DNA concentration	178.5ng/ul	160.4ng/ul	159.8ng/ul	128.7ng/ul
					A260/A280	1.848	1.836	1.856	1.827

Table 3: triton X-100 DNA kit for taking plant tissue DNA quality

Name of plant	Leaf of Maohua Bauhinian	Willow leaf	Poplar leaf	Chinese rose leaf
					Plant tissue quality	110.2mg	98.1mg	99.4mg	108.2mg
DNA concentration	354.1ng/ul	284.3ng/ul	324.9ng/ul	294.6ng/ul
					A260/A280	1.879	1.861	1.857	1.843

Example 2-three kits for Rapid extraction of DNA from three animal tissues and Yeast

As described above, 150ul (recommended by Miller manufacturer) Triton X-100 DNA lysate (either Fuile DNA lysate or Vickers DNA lysate) was placed in a 1.5ml EP tube, placed on ice, added to animal tissue, and ground thoroughly using a MY-20 grinder. Alternatively, 150ul Triton X-100 lysate was added to the centrifuged, supernatant removed yeast and thoroughly ground in a MY-10 grinder. Then, 450ul Triton X-100 DNA lysate (Fule DNA lysate or 350ul Vickers DNA lysate) was added and mixed well. Alternatively, animal tissue and centrifuged yeast cells from supernatant were removed (using MY-10 grinder), placed in an EP tube, a small amount of liquid nitrogen was added to the EP tube, and the EP tube was fully ground using MY-20 grinder under the protection of liquid nitrogen. Animal tissue and yeast DNA were extracted according to the methods described previously, and the results were determined on a biaosharp (tables 4, 5 and 6) and detected by gel electrophoresis (FIG. 2). It can be seen that the sample treated by the MY-20 grinder and the MY-10 grinder can realize the rapid extraction of genomic DNA of animals and yeast.

Table 4: quality of DNA extracted from animal tissue and yeast by scribble happiness DNA kit

Organization name	Black fish liver	Mouse liver	Mouse lung	Yeast
					Tissue quality	30.2mg	40.2mg	35.4mg	1ml(OD：0.832)
DNA concentration	105.4ng/ul	397.4ng/ul	316.3ng/ul	241.5ng/ul
					A260/A280	1.823	1.861	1.841	1.836

Table 5: extraction of animal tissue and yeast DNA quality by using Vickers DNA kit

Organization name	Black fish liver	Mouse liver	Mouse lung	Yeast
					Tissue quality	40.3mg	37.9mg	36.4mg	1ml(OD：0.832)
DNA concentration	117.4ng/ul	341.5ng/ul	261.7ng/ul	238.0ng/ul
					A260/A280	1.837	1.859	1.847	1.861

Table 6: triton X-100 DNA kit for extracting animal tissue and yeast DNA quality

Organization name	Black fish liver	Mouse liver	Mouse lung	Yeast
					Tissue quality	38.4mg	35.1mg	33.9mg	1ml(OD：0.832)
DNA concentration	256.3ng/ul	384.3ng/ul	317.9ng/ul	202.7ng/ul
					A260/A280	1.825	1.861	1.841	1.858

The result shows that the biological sample processing method provided by the invention has better universality in the aspect of biological DNA extraction, adopts a common grinding instrument in the field, is suitable for DNA extraction of various biological tissues, has higher DNA concentration and purity, and can be used for subsequent molecular biology experiments and the like. More remarkably, the method has very short operation time, and provides more possibilities for shortening the whole experiment time and improving the experiment efficiency.

Sources of reagents used in the present invention:

triton X-100 DNA kit Han Yuansheng International technology for medicine (Beijing) Ltd

Shanyi DNA kit Han Yuan Biotechnology International science and technology (Beijing) Co., Ltd

Weikele DNA kit Han remote International technology for medicine (Beijing) Ltd

Claims (10)

1. A biological tissue sample processing method for rapid extraction of DNA, characterized by comprising the steps of:

(a) adding the simply treated biological tissue into a grinding device or a grinding instrument;

(b) sufficiently grinding the biological tissue;

(c) transferring the ground sample into a DNA extraction solution for DNA extraction.

2. The biological sample processing method according to claim 1, for rapid extraction of DNA, characterized in that: the biological sample described in step (a) requires simple pre-processing such as: cutting the plant leaves into smaller leaf tissues and the like.

3. The biological sample processing method according to claim 1, for rapid extraction of DNA, characterized in that: and (b) grinding the biological tissue by using a grinder under the condition of lysate protection or grinding the biological tissue by using a grinder under the condition of liquid nitrogen refrigeration. Milling is preferably completed within 5 minutes.

4. The method for extracting plant tissue DNA according to claim 1, wherein: step (c) further comprises:

i. transferring the treated biological tissue into a first solution, uniformly mixing, standing at room temperature, and adding a corresponding second solution to some biological tissue, uniformly mixing and standing; centrifuging and retaining the supernatant;

ii, adding the second solution and mixing uniformly, adding corresponding third solution and mixing uniformly if necessary, transferring into an adsorption column, centrifuging and removing filtrate;

adding the third solution and the corresponding fourth solution if necessary into the adsorption column, centrifuging and discarding the filtrate;

fully drying the adsorption column and transferring to a new container;

and v, dropwise adding the fourth solution and the corresponding fifth solution if necessary into the center of the adsorption column, standing at room temperature, centrifuging, and collecting filtrate.

5. The method for extracting plant tissue DNA according to claim 4, wherein: and steps i to vi are all completed in a room temperature environment.

6. The method for extracting plant tissue DNA according to claim 4, wherein: the first solution is a lysis solution which is a Triton X-100 DNA lysis solution (Fuile DNA lysis solution or Vicker DNA lysis solution) containing RNase A, and the second solution of the Vicker DNA kit is 5M NaCl.

7. The method for extracting plant tissue DNA according to claim 4, wherein: the second solution is isopropanol (a third solution of a Vickers DNA kit) in a ratio of 0.6-1: 1; the third solution is rinsing liquid (fourth solution of the Vickers DNA kit) which contains 65-80% of ethanol; the fourth solution is a TE eluate or pure water (fifth solution of Vickers DNA kit).

8. A kit for carrying out the method according to any one of claims 1 to 7, and a vector describing the method according to any one of claims 1 to 7.

9. A computer readable carrier carrying a computer program comprising instructions for carrying out the method according to any one of claims 1 to 7.

10. An automated nucleic acid extraction device, the device comprising: a liquid filling/pipetting assembly, a centrifugation assembly, a grinding assembly and a controller electrically connected with the assemblies; the controller comprises the computer readable carrier of claim 9.

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