CN110592074B - Rapid algae DNA extraction method - Google Patents
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- CN110592074B CN110592074B CN201910918762.2A CN201910918762A CN110592074B CN 110592074 B CN110592074 B CN 110592074B CN 201910918762 A CN201910918762 A CN 201910918762A CN 110592074 B CN110592074 B CN 110592074B
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- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/101—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by chromatography, e.g. electrophoresis, ion-exchange, reverse phase
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
Abstract
The invention discloses a rapid algae DNA extraction method, relates to the technical field of organism DNA extraction, can efficiently and rapidly extract DNA of various algae samples, ensures that the extracted DNA has high concentration and high purity, and can be directly used for subsequent research and detection. Filtering and collecting 50mg of algae sample in a 1.5mL centrifuge tube, sequentially adding BufferA, BufferB and BufferC in sequence, and respectively oscillating and uniformly mixing; centrifuging, adding Buffer C, Buffer D and glass beads into the precipitate, oscillating and mixing uniformly, centrifuging, removing protein by phenol chloroform isoamylol after centrifuging, adding isopropanol solution, oscillating and mixing uniformly, adding the mixture into a silicon substrate membrane nucleic acid purification column, centrifuging, discarding waste liquid, rinsing, centrifuging again after rinsing, drying, and adding eluent to obtain the algae genome DNA solution.
Description
Technical Field
The invention relates to the technical field of organism DNA extraction, in particular to a rapid algae DNA extraction method.
Background
The metagenomics brings unprecedented opportunities for maximally mining microbial resources, and becomes the most important hotspot and frontier for the research and development of the international life science and technology. In order to fully recognize the abundant resources of non-culturable microorganisms, in conjunction with the development of modern molecular biology techniques, a number of new methods and techniques have been established which do not require independent cultivation of the microorganisms. Such as DGGE, 16s sequencing, metagenomic sequencing, etc. Rapid high quality environmental DNA acquisition is an important prerequisite for achieving the above-mentioned studies, and therefore, research of environmental DNA extraction methods is necessary.
Algae are currently considered to be the major factor responsible for bloom outbreaks. In order to detect and prevent cyanobacterial bloom, research on related algae toxin-producing genes, extraction of algae genome DNA for species identification and the like are indispensable steps of related research. At present, the methods of environmental DNA mainly comprise a CTAB method, a cracking method, a cellulase method and a partially commercialized kit.
Although the method for extracting the environmental DNA can be used for extracting the genomic DNA of the algae or an environmental water sample, the method also has the problems of long extraction period, complicated extraction method and complicated extraction reagent, the extraction period generally needs more than 3 hours, some commercial kits need 6-8 hours of operation, the complicated extraction method is not suitable for high-throughput research, and the complicated extraction reagent causes higher cost.
The prior published patent [ CN 104818269A ] relates to a method for extracting blue algae genome DNA, belonging to the technical field of organism DNA extraction. The invention collects 100mg algae cells in a 1.5ml Ep tube by centrifugation, 50 mul of Buffer A is added and placed in a water bath at 95 ℃ for 10min, then 50 mul of Buffer B is added and mixed evenly, and the extracted DNA sample can be directly used or stored in a refrigerator at-20 ℃. Compared with the prior art, the extraction method of the blue algae genome DNA has short extraction time of the genome DNA, and only needs about 20 min; the extraction cost of the algae genome DNA is greatly reduced, and the extraction cost is reduced by over 2/3; can extract the algae genome DNA with high flux and is used for PCR amplification, fluorescent quantitative PCR and other related molecular biology researches. However, the concentration of the extracted DNA is 0.29 mug/muL, A260/A280 is 1.546, although the concentration of the extracted genomic DNA is high, the purity is low, protein pollution exists, and the extracted genomic DNA can be used for related molecular biology research, but the existence of a small amount of protein still has certain influence on the research.
Disclosure of Invention
The invention aims to provide a rapid algae DNA extraction method, which can efficiently and rapidly extract DNA of various algae samples, enables the extracted DNA to have high concentration and high purity, and can be directly used for subsequent research and detection.
In order to achieve the purpose, the invention provides the following technical scheme: a rapid algae DNA extraction method comprises the following steps:
s1: filtering and collecting 50mg algae samples, adding 100 microliters of Buffer A and 500 microliters of sterile water into a 1.5mL centrifuge tube, uniformly mixing, adding 400 microliters of Buffer B, performing vortex oscillation for uniformly mixing for 30s, then adding 300 microliters of Buffer C, performing vortex oscillation for uniformly mixing for 30s, then centrifuging for 2 minutes at 8000g, and removing supernatant;
s2: adding 300 microliters of Buffer C and 0.5g of glass beads with the size of 0.5mm into the precipitate obtained in the step S1, performing vortex oscillation for 30S, adding 600 microliters of Buffer D, performing vortex oscillation for 1min, and centrifuging for 2min at 10000 g;
s3: adding 750 μ l of supernatant obtained from S2 into another clean centrifuge tube, adding equal volume of phenol chloroform isoamyl alcohol, vortex shaking for 30S, centrifuging at 16000g for 1min,
s4: carefully transferring the solution obtained in the step S3 to a new clean centrifugal tube, adding isopropanol with the same volume, and uniformly mixing;
s5: adding the sample obtained in S4 to a silica matrix membrane nucleic acid purification column, centrifuging at 12000g for 30S, discarding the waste liquid, adding 1mL of rinsing liquid, centrifuging at 12000g for 30S, and drying at room temperature for 2 min;
s6: adding 50-100 microliters of eluent into the sample obtained in the step S5 to obtain an algae genome DNA solution, and carrying out electrophoresis detection in 1% agarose gel;
wherein, the formulas of the Buffer A, the Buffer B, the Buffer C, the Buffer D, the rinsing solution and the eluent are as follows:
Buffer A：0.1-1.5mol/L Tris-HCl，pΗ＝3-5；
Buffer B：2-4mol/L NaOH，2％W/V Tween 20；
Buffer C：0.1-0.2mol/L Tris-HCl，pΗ＝7-9；
Buffer D：
adding double distilled water to dissolve and regulating p H to be 7-9, and fixing the volume to 10 mL;
rinsing liquid: 15-25mmol/L NaCl, L-3mmol/L Tris-HCl, p Η ═ 7.0-8.0;
eluent: 5-15mmol/L Tris-HCl, p Η ═ 8.0-9.0.
Preferably, the Buffer a, the Buffer B, the Buffer C, the Buffer D, the rinsing solution and the eluent are specifically:
Buffer A：1.0mol/L Tris-HCl，pΗ＝4.5；
Buffer B：3.0mol/L NaOH，2％W/V Tween 20；
Buffer C：0.1mol/L Tris-HCl，pΗ＝8.0；
Buffer D：
adding double distilled water to dissolve and adjusting p H to 8.0, and fixing the volume to 10 mL;
rinsing liquid: 20mmol/L NaCl, 2mmol/L Tris-HCl, p Η 7.5;
eluent: 10mmol/L Tris-HCl, p Η ═ 8.5.
Preferably, the preparation ratio of the phenol, chloroform and isoamyl alcohol is phenol: chloroform: isoamyl alcohol 25: 24: 1.
preferably, the method for extracting the algae DNA comprises pretreatment of an algae sample, and extraction and purification of algae genome DNA.
Preferably, when the algae genomic DNA extracted by the algae DNA extraction method is used for PCR or quantitative PCR reaction, 0.1% W/V bovine serum albumin is added in addition to primers, dNTP, buffer Mg2+ and enzymes necessary for PCR reaction.
Compared with the prior art, the invention has the beneficial effects that:
1. the extraction time of the algae genome DNA extraction method is short, the extraction steps are simple, the method is suitable for various algae samples, and compared with the traditional method, the used medicament is simpler in extraction, the whole algae genome DNA extraction process is controlled within 30 minutes, the extraction time of the algae genome DNA is greatly reduced, and the extraction cost is reduced by more than 2/3 by reducing the time;
2. the invention can extract the genomic DNA of algae in parallel, greatly simplifies the sample pretreatment and extraction process in the extraction of the algae DNA in water environment, has high concentration and high purity of the extracted algae DNA, can be directly used for related molecular biology researches such as PCR amplification, fluorescent quantitative PCR, high-throughput sequencing and the like, can amplify a target gene fragment of about 318bp in a 16s V3 area of the algae sample, has no non-specific amplification, and has the amplification target fragment size of about 128bp in the fluorescent quantitative PCR.
Drawings
FIG. 1 shows the result of extracting genomic DNA from a blue algae sample in lake water;
FIG. 2 shows PCR amplification of the rDNA of Microcystis 16S V3;
FIG. 3 is a qPCR amplification curve for detecting toxigenic microcystis FACHB-315 by fluorescent quantitative PCR.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments.
Example 1: algae genome DNA extraction
1. The reagent of the invention:
ribozyme-free water, manufacturer: ambion, cat #: AM9932
BSA, manufacturer: NEB, cat no: b9001
2. The invention relates to a main experimental instrument:
eppendorf high-speed refrigerated centrifuge
3. Gel imaging system, manufacturer: shanghai Qixiang scientific instruments Co Ltd
4. Performing algae genome DNA extraction
Collecting 1L of lake water samples rich in algae at different positions, filtering by using a filter membrane with the diameter of 0.22 mu m, enriching the algae samples, and extracting the algae genome DNA according to the following scheme:
1) filtering and collecting 50mg algae sample in a 1.5mL centrifuge tube, adding 100 μ L buffer A and 500 μ L sterile water, and mixing; adding 400 mu L of Buffer B, and uniformly mixing for 30s by vortex oscillation; adding 300 mu L of Buffer C, and uniformly mixing for 30s by vortex oscillation; centrifuging at 8000g for 2min (rotating speed is usually represented by rpm in low-speed centrifugation, and g in high-speed centrifugation, wherein rpm is rpm/min, and the separation factor is 1.118 times n times r squared, wherein n is rpm, and r is radius, and unit is meter), and removing supernatant;
2) adding 300. mu.L of Buffer C and 0.5g of glass beads (0.5mm) into the precipitate, performing vortex oscillation for 30s, adding 600. mu.L of Buffer D, performing vortex oscillation for 1min, centrifuging for 2min at 10000g, taking 750. mu.L of supernatant, adding the supernatant into another clean centrifugal tube, adding phenol chloroform isoamylol (phenol: chloroform: isoamyl alcohol 25: 24: 1) vortex and shake for 30s, centrifuge for 1min at 16000g, carefully transfer the supernatant to a new clean centrifuge tube, add isopropanol of equal volume, mix well, add the sample to a silica matrix membrane nucleic acid purification column, centrifuge for 30s at 12000g, discard the waste liquid, add 700 μ L of rinsing liquid, centrifuge for 30s at 12000g, dry for 2min at room temperature, add 50 μ L of eluent, centrifuge to obtain the algae genome DNA solution.
Wherein, the formulas of the Buffer A, the Buffer B, the Buffer C, the Buffer D, the rinsing solution and the eluent are as follows:
Buffer A：1.0mol/L Tris-HCl，pΗ＝4.5；
Buffer B：3.0mol/L NaOH，2％W/V Tween 20；
Buffer C：0.1mol/L Tris-HCl，pΗ＝8.0；
Buffer D：
adding double distilled water to dissolve and adjusting p H to 8.0, and fixing the volume to 10 mL;
rinsing liquid: 20mmol/L NaCl, 2mmol/L Tris-HCl, p Η 7.5;
eluent: 10mmol/L Tris-HCl, p Η ═ 8.5.
5. And (3) detecting the DNA concentration and purity:
respectively taking 2 mu L of DNA of the algae genome, measuring A260 and A280 by using a micro ultraviolet spectrophotometer (NanoDrop ND1000), and recording the concentration and purity value of the DNA;
another 5. mu.L of the algae DNA solution was electrophoretically detected in 1% agarose gel, and the detection pattern was recorded by photographing.
The detection result shows that the concentration of the 4 genomic DNAs extracted by the method is 284 ng/muL, 186 ng/muL, 178 ng/muL, 275 ng/muL and A260/A280 is 1.746, 1.694, 1.706 and 1.803 respectively, the concentration and the purity of the genomic DNAs are high, the research of subsequent experiments is achieved, and the purity is higher than that extracted by the existing extraction method.
Lanes 1-4 in the electropherogram are the extraction results of four algae samples respectively, M is molecular weight indicator (DL2000, the sizes of the bands from top to bottom are 2000bp,1000bp,750bp,500bp,250bp and 100bp respectively), and it can be seen that clear genomic DNA bands are obtained from all four algae samples, the sizes are relatively complete, the degradation is basically avoided, and the expected effect is achieved.
Example 2: microcystis 16S rDNA PCR amplification
1. The reagent of the invention:
ribozyme-free water, manufacturer: ambion, cat #: AM9932
BSA, manufacturer: NEB, cat no: b9001
PCR amplification reagents, manufacturer: baozbio corporation, cat #: R045Q
2. The experimental instrument of the invention:
eppendorf high-speed refrigerated centrifuge
3. Gel imaging system, manufacturer: shanghai Qixiang scientific instruments Co Ltd
4. Microcystis 16S rDNA PCR amplification
Performing 16S rDNA PCR amplification on the algae genome by using 16S V3 region primers, wherein the primer sequences are 16S-F: CCTACGGGNGGCWGCAG, 16S-R: GACTACHVGGGTATCTAATCC, the size of the amplified target fragment is about 318 bp.
The PCR reaction system is as follows: 10 μ M of each 1 μ L of 16s-F and 16s-R primers; 10 × PCR buffer 2.5 μ L; 25 μ M dNTP 2 μ L; MgCl21.5 μ L15 μ M; algal genomic DNA 1. mu.L; 2.5. mu.L of 0.1% W/V Bovine Serum Albumin (BSA); taq DNA polymerase, 0.5U, dd H2O made up 25. mu.L of the reaction.
The thermal cycling parameters of the PCR amplification reaction were: pre-denaturation at 94 ℃ for 2min, denaturation at 94 ℃ for 30s, annealing at 58 ℃ for l min, extension at 72 ℃ for 30s, 35 cycles, extension at 72 ℃ for 8min, and heat preservation at 10 ℃.
mu.L of the PCR product was electrophoresed on a 2% agarose gel, and the results are shown in FIG. 2. The target gene fragment of about 318bp is successfully amplified in the 16s V3 area of all four algae samples, and non-specific amplification is not carried out.
Lanes 1-4 show the amplification results of four samples, respectively, and M is the molecular weight marker DL1000 (the brightest band is 400bp), and it can be seen from the figure that the DNA extracted by the method can be directly amplified for 16s rDNA without further purification.
Example 3: fluorescence quantitative PCR detection of toxigenic microcystis FACHB-315
1. The reagent of the invention:
ribozyme-free water, manufacturer: ambion, cat #: AM9932
BSA, manufacturer: NEB, cat no: b9001
Fluorescent quantitative PCR amplification reagent, manufacturer: baozbio corporation, cat #: DRR041S
2. The experimental instrument of the invention:
eppendorf high-speed refrigerated centrifuge
Fluorescent quantitative PCR instrument, manufacturer model: thermofeisher ABI7500 quantitative PCR instrument
3. Fluorescent quantitative PCR detection
PCR amplification is carried out by using microcystin toxin-producing gene mcyE/ndaF primer,
primer sequence mcy-F: AATAAATCATAATTTAGAACSGGVGATTTAGG, respectively;
mcy-R: AATAAATCATAACGRVADTTGRTATTCAATTTCT, and the size of the amplified target fragment is about 128 bp.
The PCR reaction system is as follows: mu.M mcy-F and mcy-R primers, 1. mu.L each, 10. mu.L 2 XSSYBR Premix Ex TaqTM (TaKaRa Code: DRR041S), 1. mu.L algae genomic DNA, 2.5. mu.L 0.1% W/V Bovine Serum Albumin (BSA), dd H2O 2. mu.L, 20. mu.L total.
The PCR amplification procedure was: pre-denaturation at 94 ℃ for 2min, denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 1min, extension at 72 ℃ for 30s, 35 cycles, extension at 72 ℃ for 8min, and heat preservation at 10 ℃.
qPCR amplification was performed on an ABI7500 quantitative PCR instrument, fluorescence was collected during annealing, and each sample experiment was repeated 3 times.
Melting curve process: 95 ℃ for 1min, 65 ℃ for 1min, the temperature is increased by 0.5 ℃ from 65 ℃ every 30s, the finishing temperature is 95 ℃, and the reaction is stored at 4 ℃ after finishing the reaction.
The toxigenic microcystis is detected by fluorescent quantitative PCR, which is shown in figure 3. The reaction system added with Bovine Serum Albumin (BSA) successfully performs fluorescent quantitative PCR reaction, detects the toxigenic microcystis FACHB-315, and shows that the algae genome DNA obtained by the method can be used for fluorescent quantitative PCR.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Claims (5)
1. A rapid algae DNA extraction method is characterized by comprising the following steps:
s1: filtering and collecting 50mg algae samples, adding 100 microliters of Buffer A and 500 microliters of sterile water into a 1.5mL centrifuge tube, uniformly mixing, adding 400 microliters of Buffer B, performing vortex oscillation for uniformly mixing for 30s, then adding 300 microliters of Buffer C, performing vortex oscillation for uniformly mixing for 30s, then centrifuging for 2 minutes at 8000g, and removing supernatant;
s2: adding 300 microliters of Buffer C and 0.5g of glass beads with the size of 0.5mm into the precipitate obtained in the step S1, performing vortex oscillation for 30S, adding 600 microliters of Buffer D, performing vortex oscillation for 1min, and centrifuging for 2min at 10000 g;
s3: adding 750 μ l of supernatant obtained from S2 into another clean centrifuge tube, adding equal volume of phenol chloroform isoamyl alcohol, vortex shaking for 30S, centrifuging at 16000g for 1min,
s4: carefully transferring the solution obtained in the step S3 to a new clean centrifugal tube, adding isopropanol with the same volume, and uniformly mixing;
s5: adding the sample obtained in S4 to a silica matrix membrane nucleic acid purification column, centrifuging at 12000g for 30S, discarding the waste liquid, adding 1mL of rinsing liquid, centrifuging at 12000g for 30S, and drying at room temperature for 2 min;
s6: adding 50-100 microliters of eluent into the sample obtained in the step S5 to obtain an algae genome DNA solution, and carrying out electrophoresis detection in 1% agarose gel;
wherein, the formulas of the Buffer A, the Buffer B, the Buffer C, the Buffer D, the rinsing solution and the eluent are as follows:
Buffer A：0.1-1.5mol/L Tris-HCl，pΗ＝3-5；
Buffer B：2-4mol/L NaOH，2％W/V Tween 20；
Buffer C：0.1-0.2mol/L Tris-HCl，pΗ＝7-9；
adding double distilled water to dissolve and regulating p H to be 7-9, and fixing the volume to 10 mL;
rinsing liquid: 15-25mmol/L NaCl, L-3mmol/L Tris-HCl, p Η ═ 7.0-8.0;
eluent: 5-15mmol/L Tris-HCl, p Η ═ 8.0-9.0.
2. The rapid extraction method of algae DNA according to claim 1, wherein: the Buffer A, the Buffer B, the Buffer C, the Buffer D, the rinsing solution and the eluent are specifically as follows:
Buffer A：1.0mol/L Tris-HCl，pΗ＝4.5；
Buffer B：3.0mol/L NaOH，2％W/V Tween 20；
Buffer C：0.1mol/L Tris-HCl，pΗ＝8.0；
adding double distilled water to dissolve and adjusting p H to 8.0, and fixing the volume to 10 mL;
rinsing liquid: 20mmol/L NaCl, 2mmol/L Tris-HCl, p Η 7.5;
eluent: 10mmol/L Tris-HCl, p Η ═ 8.5.
3. The rapid extraction method of algae DNA according to claim 1, wherein: the preparation ratio of the phenol chloroform isoamyl alcohol is phenol: chloroform: isoamyl alcohol 25: 24: 1.
4. the rapid extraction method of algae DNA according to claim 1, wherein: the method for extracting the algae DNA comprises the steps of pretreatment of an algae sample, and extraction and purification of algae genome DNA.
5. The rapid extraction method of algae DNA according to claim 1, wherein: when the algae genome DNA extracted by the algae DNA extraction method is used for conventional PCR or quantitative PCR reaction, 0.1% W/V bovine serum albumin is required to be added besides primers, dNTP, buffer Mg2+ and enzyme required by the PCR reaction.
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Citations (1)
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| CN104818269A (en) * | 2015-05-20 | 2015-08-05 | 湖州师范学院 | Method for extracting genome DNA of blue algae |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104818269A (en) * | 2015-05-20 | 2015-08-05 | 湖州师范学院 | Method for extracting genome DNA of blue algae |
Non-Patent Citations (4)
| Title |
|---|
| 3种小球藻DNA提取方法的比较;陈颖 等;《植物生理学通讯》;20040108;第37卷(第3期);第242-244页 * |
| 海洋单细胞四爿藻基因组DNA的微量提取;罗立明 等;《武汉植物学研究》;20031107;第21卷(第4期);第295-300页 * |
| 金藻基因组DNA的提取与PCR扩增;杨泽民 等;《生物学杂志》;20080504;第25卷(第1期);第60-63页 * |
| 高效海藻DNA提取方法研究;韩静 等;《上海水产大学学报》;20081201;第17卷(第5期);第530-534页 * |
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