CN112575061A - Method for extracting tissue DNA by one-step method - Google Patents

Method for extracting tissue DNA by one-step method Download PDF

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CN112575061A
CN112575061A CN202011570847.5A CN202011570847A CN112575061A CN 112575061 A CN112575061 A CN 112575061A CN 202011570847 A CN202011570847 A CN 202011570847A CN 112575061 A CN112575061 A CN 112575061A
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tissue
tissue dna
extraction
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夏水秀
方坛芬
丁威
韩蓉
薛满
孙万平
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Suzhou University
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    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

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Abstract

The invention belongs to the technical field of nucleic acid extraction, and particularly relates to a method for extracting tissue DNA by a rapid one-step method, which comprises the steps of adding a tissue into a rapid one-step nucleic acid extraction reagent, standing, and completing extraction of the tissue DNA; the rapid one-step nucleic acid extraction reagent comprises the following raw materials: sodium hydroxide (NaOH), disodium ethylenediaminetetraacetate (Na)2EDTA), potassium acetate (KAc). By adopting the extraction method, the tissue only needs to be kept stand for 2-6 min with the reagent, multi-step reaction and experimental equipment are not needed, and the concentration and the purity of the extracted tissue DNA are good; and (3) standing the supernatants of 5 different time points with 2-6 points, and when the supernatants are diluted by 10 times to be used as a PCR amplification template, the electrophoresis bands of the amplification products are clear. Therefore, the method of the present invention is of great importance in point-of-care testing (POCT) of nucleic acids in tissues in situ.

Description

Method for extracting tissue DNA by one-step method
Technical Field
The invention belongs to the technical field of nucleic acid extraction, and particularly relates to a method for extracting tissue DNA by a rapid one-step method.
Background
There are many methods for extracting tissue genome DNA, and the classical methods include phenol extraction, formamide cleavage, isopropanol precipitation, guanidine hydrochloride cleavage glass rod winding, and phenol-chloroform (see Samsubuke J, Frizi EF, Mannich Abies T. molecular cloning, 2 nd edition, scientific Press, 1992: 463 and 469.), which can obtain high quality DNA, but the extraction process is complicated, and the technical and equipment requirements are high and the time is long. The salting-out method, the kit extraction method and the particle adsorption method appear through improvement and optimization on the classical DNA extraction method, but special equipment (such as a high-speed refrigerated centrifuge) is still required and the cost is high. Therefore, the preparation of a DNA fast extraction solution is the first step to realize POCT. Although some rapid one-step extracting solutions in the current market shorten the extracting time, the purity of the extracted DNA is poor, and the subsequent amplification reaction can be influenced; the step of high-temperature heating is needed, and the method is not suitable for POCT and other problems. Therefore, on-site rapid DNA extraction is a prerequisite for establishing a nucleic acid on-site detection (POCT) method.
Disclosure of Invention
The research mainly develops a rapid one-step extracting solution to rapidly extract the tissue DNA, provides a tissue DNA extracting method through extracting time and sample concentration, and simultaneously compares the tissue DNA extracting method with 2 commercial kits and 2 commercial one-step extracting solutions, so that the extracting method provided by the invention is good in effect and short in required time, and meets the requirements of POCT.
The invention adopts the following technical scheme:
a method for extracting tissue DNA by a rapid one-step method comprises the following steps of adding tissue into a rapid one-step nucleic acid extraction reagent, standing, and finishing the extraction of tissue DNA.
A method for tissue DNA amplification comprises adding tissue into a rapid one-step nucleic acid extraction reagent, standing, and collecting supernatant; and (3) performing PCR reaction by using the supernatant diluent as a template to complete the amplification of the tissue DNA.
The rapid one-step nucleic acid extraction reagent consists of sodium hydroxide, disodium ethylene diamine tetraacetate, potassium acetate and water; wherein the concentration of the sodium hydroxide is 0.5 mol/L, the concentration of the ethylene diamine tetraacetic acid is 10 mmol/L, and the concentration of the potassium acetate is 3 mol/L; adding sodium hydroxide, disodium ethylene diamine tetraacetate and potassium acetate into water to form the rapid one-step nucleic acid extraction reagent.
In the invention, the standing time is 1-6 min, preferably 2-5 min, and more preferably 2 min. The method can reach the simple and rapid standard, and the whole extraction process is less than 5min when the DNA one-step extracting solution is used for extracting the tissue DNA.
In the invention, the supernatant is diluted and used as a template for PCR reaction to complete the amplification of tissue DNA; preferably, the dilution factor is 10 fold. The methods for taking and diluting the supernatant are conventional techniques, and the subsequent PCR reaction and electrophoresis test are also conventional methods.
The creativity of the invention lies in providing a new extraction method, which can complete the extraction of tissue DNA within 5 minutes, and after dilution, the tissue DNA can be used as a template for amplification, and the invention has the advantages of clear amplification result, high concentration of extracting solution and good purity.
Drawings
FIG. 1 is a photograph of the electrophoresis of the DNA template after PCR amplification, note: lanes 1-6 are standing for 1, 2, 3, 4, 5, 6min (stock solution is template), respectively; lanes 7-12 are standing for 1, 2, 3, 4, 5, 6min (10 times diluted as template); 13 as positive control (TIANGEN extracting DNA stock solution); and 14 is a negative control.
Detailed Description
The tissue is added into a rapid one-step nucleic acid extraction reagent, standing is carried out, and the tissue DNA can be extracted by taking the supernatant conventionally without other steps; the resulting supernatant was diluted and used as an amplification template for DNA amplification.
Experimental Material
Main instrument
Figure 303889DEST_PATH_IMAGE001
Main reagent consumable
Figure 440472DEST_PATH_IMAGE002
Example A DNA extraction
A rapid one-step nucleic acid extraction reagent is prepared from sodium hydroxide (NaOH), disodium ethylene diamine tetraacetate (Na 2 EDTA), potassium acetate (KAc) and water by adding sodium hydroxide, disodium ethylene diamine tetraacetate and potassium acetate into water, and conventionally mixing to obtain a rapid one-step nucleic acid extraction reagent, namely a one-step DNA extraction reagent; wherein the concentration of the sodium hydroxide is 0.5 mol/L, the concentration of the ethylene diamine tetraacetic acid is 10 mmol/L, and the concentration of the potassium acetate is 3 mol/L.
Weighing 20mg of pork liver sample, cutting into sesame grain size, adding 200 μ L of the above one-step DNA extraction reagent, standing for 2min, and sucking supernatant conventionally; the supernatant was diluted 10-fold with water as a template for PCR amplification in less than 5 minutes.
Comparative example 1
Adding sodium hydroxide and disodium ethylene diamine tetraacetate into water to form a one-step DNA extraction reagent, wherein the concentration of the sodium hydroxide is 0.5 mol/L, and the concentration of the disodium ethylene diamine tetraacetate is 10 mmol/L. Weighing 20mg of pork liver sample, cutting into sesame grain size, adding 200 μ L of the above extraction reagent, standing for 2min, and sucking supernatant conventionally.
Comparative example No. two
Adding sodium hydroxide, disodium ethylene diamine tetraacetate and potassium acetate into water to form a one-step DNA extraction reagent, wherein the concentration of the sodium hydroxide is 0.5 mol/L, the concentration of the disodium ethylene diamine tetraacetate is 10 mmol/L, and the concentration of the potassium acetate is 5 mol/L. Weighing 20mg of pork liver sample, cutting into sesame grain size, adding 200 μ L of the above extraction reagent, standing for 2min, and sucking supernatant conventionally.
Weighing 20mg of a pork liver sample, cutting into sesame grains, and performing DNA extraction according to the two purchased kits and the two steps of one-step rapid extraction reagents to obtain supernatant.
Determination of concentration and purity
Detecting the DNA of the pig liver extracted by the 7 methods by using a Nanodrop2000c nucleic acid protein instrument to obtain the DNA concentration and A260 nm/A280 nmA ratio. The specific data are shown in Table 1.
Figure 781455DEST_PATH_IMAGE003
As can be seen from Table 1, the extraction concentration and purity (A260/A280) of the extraction solution and the TIANGEN tissue genome extraction kit are good, but the TIANGEN tissue genome extraction kit needs long time, needs about 2 hours and is not suitable for rapid POCT, and the extraction solution can complete DNA extraction within 5 min. In terms of test cost, the cost of the extracting solution is far lower than that of the kit. In the aspect of auxiliary equipment, the method does not need auxiliary equipment, the supernatant can be obtained after standing for 2min at room temperature, and the operation process of the TIANGEN tissue genome extraction kit needs heating, high-speed centrifugation and other steps; the columnar animal mitochondria DNAout has good extraction purity of PCR grade (Beijing Tian Enze), but has lower concentration; the extraction concentration of two commercial general one-step DNA extracting solutions is higher, but the two methods both need high-temperature heating and long time, are not as good as the extraction method of the invention carried out at room temperature, and are suitable for POCT. The results of the first and second comparative examples show that the extraction concentration and purity of the self-prepared one-step extracting solution are remarkably improved.
Example two
Weighing 20mg of a pork liver sample, cutting into sesame grains, adding 200 mu L of the one-step DNA extraction reagent in the embodiment I, standing, and taking a supernatant; timing from the addition of the extraction reagent, respectively taking and placing sample supernatants after standing for 1, 2, 3, 4, 5 and 6min in a new 1.5ml centrifuge tube; the supernatant was diluted 10 times and the undiluted supernatant was used as DNA templates for PCR amplification and electrophoresis. The results of the experiment are shown in FIG. 1.
Weighing 20mg of a pork liver sample, cutting into sesame grain size, extracting DNA by adopting the conventional kit, and performing the same PCR amplification and electrophoresis by using the DNA template. The results of the experiment are shown in FIG. 1.
Preparing a PCR reaction system:
Figure 712502DEST_PATH_IMAGE004
Figure 903049DEST_PATH_IMAGE005
PCR reaction procedure:
Figure 843323DEST_PATH_IMAGE006
agarose gel electrophoresis detection
The agar plate is placed into an electrophoresis cell and is tightly attached to the edge, samples are sequentially injected with 4.5 mu L, and the samples are injected with 1.5 mu L mark (100 bp). Electrophoresis procedure: u =140V I =200mA time =30 min.
Gel imaging
Placing the agar plate in a gel imager, enabling the part for electrophoresis sample running to be in the middle → starting a gel imaging system → starting a 300nm light source, adjusting focusing, zooming and aperture, making an image clear → scanning the image → closing the light source, saving the image, and taking out the agar plate.
The result of FIG. 1 shows that when the DNA extraction reagent of the embodiment is used for one-step extraction, the undiluted template cannot be effectively amplified, when the diluted template is 10 times, the electrophoresis bands of the amplified products of the extracted DNA at 5 different times of standing for 2-6 min are clear, and are equivalent to the 13 lane bands, and the electrophoresis band is darker when the template is standing for 1 min. And comparing results, selecting the self-prepared one-step extracting solution, standing for 2-5 min, and diluting the supernatant by 10 times to be used as a PCR amplification template.
The research uses pork liver as experimental material, selects the extracting solution of the invention to match with the extraction method, and compares the extracting quality of DNA with commercial kits and general one-step extraction reagents in the market, and the experimental result shows that the whole extraction process only needs less than 5min by using the extraction method of the invention, the extracted DNA can meet the requirement of PCR amplification, and the method reaches the standard of simple, rapid and on-site extraction.
Sequence listing
<110> Suzhou university
<120> method for extracting tissue DNA by rapid one-step method
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
actactcata cccagcaagc 20
<210> 2
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
taaggtgaca ataggtagtc ct 22

Claims (10)

1. A method for extracting tissue DNA by a rapid one-step method is characterized by comprising the following steps of adding tissue into a rapid one-step nucleic acid extraction reagent, standing, and finishing the extraction of the tissue DNA; the rapid one-step nucleic acid extraction reagent comprises sodium hydroxide, disodium ethylene diamine tetraacetate and potassium acetate.
2. The method for rapid one-step extraction of tissue DNA according to claim 1, wherein the rapid one-step nucleic acid extraction reagent is prepared by adding sodium hydroxide, disodium edetate and potassium acetate into water.
3. The method for rapid one-step extraction of tissue DNA according to claim 2, wherein the concentration of sodium hydroxide is 0.5 mol/L; the concentration of the ethylene diamine tetraacetic acid disodium is 10 mmol/L; the concentration of potassium acetate is 3 mol/L.
4. The method for rapid one-step extraction of tissue DNA according to claim 1, wherein the tissue DNA is extracted by taking the supernatant after standing to obtain the tissue DNA.
5. The method for extracting tissue DNA by the rapid one-step method according to claim 1, wherein the standing time is 1-6 min.
6. The method for rapid one-step extraction of tissue DNA according to claim 5, wherein the standing time is 2-5 min.
7. A method for tissue DNA amplification is characterized by comprising the following steps of adding tissue into a rapid one-step nucleic acid extraction reagent, standing, and taking supernatant; and (3) performing PCR reaction by using the supernatant as a template to complete the amplification of the tissue DNA.
8. The method for tissue DNA amplification according to claim 7, wherein the tissue is porcine liver.
9. The method of claim 7, wherein the tissue DNA is amplified by diluting the supernatant and performing PCR reaction using the diluted supernatant as a template.
10. The method for tissue DNA amplification according to claim 7, wherein the rapid one-step nucleic acid extraction reagent is prepared by adding sodium hydroxide, disodium edetate and potassium acetate to water.
CN202011570847.5A 2020-12-26 2020-12-26 Method for extracting tissue DNA by one-step method Pending CN112575061A (en)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109468311A (en) * 2018-11-27 2019-03-15 河南普诺易生物制品研究院有限公司 A method of extracting the cellular lysate liquid, kit and extraction Plasmid DNA of Plasmid DNA

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109468311A (en) * 2018-11-27 2019-03-15 河南普诺易生物制品研究院有限公司 A method of extracting the cellular lysate liquid, kit and extraction Plasmid DNA of Plasmid DNA

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
林海等: "《环境工程微生物学实验教程》", 31 October 2020, 冶金工业出版社 *

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