CN102643800A - Method for extracting plant deoxyribonucleic acid (DNA) and special kit thereof - Google Patents

Abstract

The object of the present invention is to provide a plant DNA extraction method called mCTAB and a special kit thereof. The test kit provided by the present invention comprises buffer A, and described buffer A is made up of solute and solvent; Described solute and its final concentration in described buffer A are as follows: disodium edetate 4-6mmol ·L ^-1 , NaCl 0.2-0.3M, polyvinylpyrrolidone 1-3g/100ml; 1M Tris-HCl buffer solution with pH 8.0 8-12ml/100ml; the solvent is water. The mCTAB method of the present invention for extracting plant DNA has five advantages of high yield, good quality, high success rate of PCR amplification, low cost, and good versatility, and can meet the needs of DNA extraction in general molecular biology research, and is especially suitable for scientific research funds. Abundant researchers and extraction of large quantities of plant DNA.

Description

A method for extracting plant DNA and its special kit

technical field

The invention relates to a method for extracting plant DNA and a special kit thereof.

Background technique

DNA is the basic genetic material of plants and the carrier of plant genetic information. A certain amount of high-quality DNA samples are the basis for molecular biology research such as restriction enzyme digestion, PCR amplification, molecular hybridization, genetic polymorphism analysis, and genomics. Therefore, how to obtain a certain amount and high-quality DNA samples is extremely important.

Plant cells have cell walls, which contain more secondary metabolites such as polysaccharides, and the types and contents of secondary metabolites in different plants vary greatly, and sometimes the types and contents of secondary metabolites in different organs or tissues of the same plant are also different. Similarly, it is difficult to obtain high-quality DNA. A DNA extraction method optimized for one particular species may not necessarily work for another. The traditional CTAB method is currently the most widely used DNA extraction method, but due to the differences in the chemical composition and tissue structure of plant materials, the extraction effect of the traditional CTAB method is sometimes not good, and its use is limited. Therefore, there is an urgent need for a simple, efficient, economical and versatile plant DNA extraction method.

Contents of the invention

One object of the present invention is to provide a method for extracting plant DNA and a special kit thereof.

The kit for extracting plant DNA provided by the present invention comprises buffer A; said buffer A is made up of solute and solvent; said solute and its final concentration in said buffer A are as follows: ethylenediamine Disodium tetraacetate 4-6mmol·L ^-1 , NaCl 0.2-0.3M, polyvinylpyrrolidone 1-3g/100ml; Tris-HCl buffer solution with a concentration of 1M and a pH of 8.0 8-12ml/100ml; the solvent for water.

Buffer B may also be included in the above kit; the buffer B is composed of a solute and a solvent: the solute and its final concentration in the buffer B are as follows: Tris-HCl with a concentration of 1M and a pH of 8.0 Buffer 8-12ml/100ml, disodium edetate 20-30mmol·L ^-1 , NaCl 1.2-1.6M, CTAB2.5-3.5g/100ml, sodium metabisulfite 0.5-1.5g/100ml, Sodium ascorbate 0.5-1.5g/100ml, polyvinylpyrrolidone 1-3g/100ml, β-mercaptoethanol 80-120ul/100ml; the solvent is water.

In any of the above-mentioned kits, the buffer A can specifically be as follows: the solute and its final concentration in the buffer A are as follows: disodium edetate 5 mmol·L ^-1 , NaCl 0.25M, polyvinylpyrrolidone 2g/100ml; Tris-HCl buffer solution with a concentration of 1M and a pH of 8.0 10ml/100ml;

In any of the above kits, the buffer B can specifically be as follows: the solute and its final concentration in the buffer B are as follows: 10ml of Tris-HCl buffer solution with a concentration of 1M and a pH of 8.0 /100ml, disodium edetate 25mmol·L ^-1 , NaCl 1.4M, CTAB 3g/100ml, sodium metabisulfite 1g/100ml, sodium ascorbate 1g/100ml, polyvinylpyrrolidone 2g/100ml, β- Mercaptoethanol 100ul/100ml.

The method for extracting plant DNA provided by the present invention comprises the following steps:

(1) Mix the buffer solution A with the plant tissue powder, ice bath, centrifuge, discard the supernatant, and collect the precipitate;

(2) Mix buffer B with the precipitate obtained in step (1), place, centrifuge, and collect the supernatant;

(3) DNA is extracted from the supernatant obtained in step (2) with chloroform isoamyl alcohol solution, and then the steps of removing RNA and precipitating DNA are carried out to obtain the target DNA;

The buffer A and the buffer B are both the buffer A and the buffer B in any of the above-mentioned kits.

In the above method, in the step (1), the time of the ice bath is 10min-20min.

In the above method, in the step (2), the placing method is a water bath at 60°C-70°C for 90-120min, specifically a water bath at 65°C for 100min.

In the above method, after the step (1) and before the step (2), the following repeated steps are also included: mixing the precipitate obtained in the previous step with the buffer A, ice bathing, centrifuging, and discarding the supernatant , collect the precipitate; until the supernatant is not viscous.

In the above method, in the step (1), the ratio of buffer A to plant tissue powder is 1 mL of buffer A: 20 mg of plant tissue powder; invert and mix 2-3 times during the ice bath; Described centrifugation is 7000 × g centrifugation 10min; The time of described ice bath is 15min;

In the above method, in the step (2), the ratio of the buffer B to the plant tissue powder is 0.7mL buffer B: 20mg plant tissue powder; invert and mix several times during the water bath; The centrifugation is 10000×g for 10 min.

In the above method, in the step (3), the method for extracting DNA from the supernatant obtained in the step (2) with the chloroform isoamyl alcohol solution is as follows: add 0.7mL to the supernatant obtained in the step (2). Chloroform isoamyl alcohol solution, mixed upside down for 10min, 10000 × g, centrifuged for 10min, and collected the supernatant; in the chloroform isoamyl alcohol solution, the volume ratio of chloroform: isoamyl alcohol was 24:1; the above-mentioned chloroform isoamyl alcohol step was repeated until After centrifugation, there is no precipitation between the two liquid surfaces; then add 0.5mL isopropanol, mix well, place at -20°C for 20min, centrifuge at 10000×g for 10min, and discard the supernatant;

In the above method, in the step (3), the method for removing RNA includes the following steps: adding 0.1 mL of RNase solution with a concentration of 100 mg·L ⁻¹ , placing it at 37° C. for 30-60 min; adding 0.1 mL of deionized water , 0.1mL 5M NaCl solution, 0.8mL 95% absolute ethanol, mix well, centrifuge at 10000×g for 10min, discard the supernatant;

In the above method, in the step (3), the method for precipitating DNA is as follows: add 0.5mL 75% aqueous ethanol solution, bounce the precipitate, centrifuge at 10000 × g for 2min, and discard the supernatant.

In the above method, the plant tissue is a plant leaf.

In the above method, the plant is any one of the following: Phyllostachys brevifolia, fern, Pinus tabulaeformis, Erqiao Magnolia, grapes, Chinese rose, amaranthus retroflexus, beautiful sunflower, bamboo, Tibetan tiger head orchid .

The mCTAB method of the present invention for extracting plant DNA has five advantages of high yield, good quality, high success rate of PCR amplification, low cost, and good versatility, and can meet the needs of DNA extraction in general molecular biology research, and is especially suitable for scientific research funds. Abundant researchers and extraction of large quantities of plant DNA.

Description of drawings

Fig. 1 is the DNA agarose gel electrophoresis detection result of 10 kinds of plants extracted by five methods. The concentrations of λDNA are 10ng, 25ng and 50ng from left to right, respectively. Numbers 1-10 are sample numbers (same as Table 1). I, II, III, and IV represent DNA extraction kits from four companies, respectively.

Fig. 2 is the situation of the DNA amplification of four commonly used fragments of DNA barcodes from 10 kinds of plants extracted by five methods. Primer conditions: A.rbcLaf/r; B.trnH/psbA; C.matK472F/1248R; D.ITS1/ITS4. Numbers 1-10 are sample numbers (same as Table 1); MD is mCTAB; I, II, III and IV represent the DNA extraction kits of the four companies respectively.

Detailed ways

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

The raw materials used in each of the following embodiments and comparative examples are as follows: select 10 common plants (Table 1), respectively as bryophytes, ferns, gymnosperms, basal angiosperms, monocots, roses, chrysanthemums Representatives of major clades such as plants (APG III, 2009). These 10 kinds of plants also include different growth habits such as trees, shrubs, vines, perennial herbs, and annual herbs, which are representative. The plant samples were all collected from the Botanical Garden of the Institute of Botany, Chinese Academy of Sciences, and the digital image information and voucher specimens were deposited in the Herbarium (PE) of the Institute of Botany, Chinese Academy of Sciences. The fresh leaves of the 10 plant materials were collected, dried at 40°C and preserved with silica gel for later use.

Table 1. Materials used for comparative studies.

The detection method to the productive rate and quality of DNA in following embodiment and comparative example is as follows:

(1) Electrophoresis detection

The fragment size, degradation and concentration of the DNA extracted by the five DNA extraction methods were detected by 1% agarose gel electrophoresis. Take 1 μL of DNA stock solution, add 9 μL of electrophoresis buffer, mix well, point into 1% agarose gel, electrophoresis at 5V/cm for 30min, observe and take pictures on a UV gel imager. Using λDNA at three concentrations of 10ng, 25ng and 50ng as a standard, Quantity One (BIO-RAD) calculates the amount of DNA, thereby estimating the DNA yield.

(2) Spectrophotometer detection

Take 1 μL of DNA stock solution, and measure the OD value and A260/280 ratio of DNA extracted by five DNA extraction methods with a NanoDrop 2000 micro-spectrophotometer in the United States. The yield of DNA was calculated according to the OD value of DNA determined by spectrophotometer.

(3) PCR detection

The DNA stock solutions extracted by the five DNA extraction methods were diluted to 50ng/μL, and then PCR amplification was carried out with plant DNA barcode candidate fragments (Kress et al., 2005; Plant Working Group 2009; Li et al.2011), and the primers used were : rbcLaf/r (Kress et al., 2007), trnH/psbA (Kress et al., 2005), matK472F/1248R (Yu et al., 2011) and ITS1/ITS4 (Henrion et al., 1992).

PCR amplification uses 10 μL reaction system, including 10×PCR Buffer (containing MgCl ₂ ) 1 μL, 2mmol L ^-1 dNTPs 1 μL, 5 μmol L ^-1 Primer F/R 0.5 μL, 0.5U Taq DNA polymerase 0.1Ml, Template DNA 1 μL, ddH ₂ O 5.9 μL.

The PCR amplification reaction was completed on the BIO-RAD C1000TM Thermal Cycler PCR instrument. The PCR amplification program was pre-denaturation at 94°C for 4 min; denaturation at 94°C for 30 s; annealing at 52°C for 40 s; extension at 72°C for 1 min; 35 cycles; and finally extension at 72°C for 10 min. The PCR products were electrophoresed on 1% agarose gel to detect the success of DNA amplification, and the ultraviolet gel imager was observed and photographed.

Embodiment 1, the method for extracting plant DNA of the present invention (hereinafter referred to as mCTAB method)

1. Weigh 20mg of plant dry material, add quartz sand, grind it into powder, and transfer the powder to a 2.0mL centrifuge tube.

2. Add 1mL of pre-cooled buffer A, mix well and then ice-bath for 15min; invert and mix 2-3 times during the ice-bath. Centrifuge at 7000×g for 10 min and discard the supernatant.

3. Repeat step 2 until the supernatant is not viscous.

4. Add 0.7mL buffer B (Table 3), mix well and then bathe in 65°C water bath for 90-120min, invert and mix several times during the water bath process. Centrifuge at 10000×g for 10min, and aspirate the supernatant into a new 2.0mL centrifuge tube.

5. Add 0.7 mL of chloroform isoamyl alcohol solution (chloroform: isoamyl alcohol = 24:1), invert and mix for 10 min. Centrifuge at 10000×g for 10min, and aspirate the supernatant into a new 1.5mL centrifuge tube.

6. Repeat step 5 until there is no sediment between the two liquid surfaces after centrifugation.

7. Add 0.5mL pre-cooled isopropanol, mix gently, and place at -20°C for 20min. Centrifuge at 10000×g for 10 minutes, and discard the supernatant.

8. Add 0.1 mL of RNase (100 mg·L ^-1 ), and place at 37°C for 30-60 min. Add 0.1mL deionized water, 0.1mL 5M NaCl, 0.8mL pre-cooled 95% absolute ethanol, and mix gently. Centrifuge at 10000×g for 10 min, discard the supernatant.

9. Add 0.5mL 75% ethanol, gently bounce the precipitate, centrifuge at 10000×g for 2min, and discard the supernatant.

10. Repeat step 9.

11. Air-dry ethanol, add 0.1mLTE to dissolve DNA.

Buffer A consists of the following solutes and solvents:

The solute and its final concentration in buffer A are as follows: disodium edetate 5mmol·L ^-1 , NaCl 0.25M, polyvinylpyrrolidone 2g/100ml; 1M Tris-HCl buffer with pH 8.0 10ml /100ml; solvent is water.

Buffer B consists of the following solutes and solvents:

The solute and its final concentration in buffer B are as follows: 1M Tris-HCl buffer solution with pH=8.0 10ml/100ml, disodium edetate 25mmol·L ^-1 , NaCl 1.4M, CTAB 3g/100ml, Sodium metabisulfite 1g/100ml, sodium ascorbate 1g/100ml, polyvinylpyrrolidone 2g/100ml, β-mercaptoethanol 100ul/100ml. The solvent is water.

Embodiment 2,

The method is basically the same as in Example 1, the differences are as follows:

Buffer A is composed of solute and solvent; the solute and its final concentration in buffer A are as follows: disodium edetate 4mmol·L ^-1 , NaCl 0.2M, polyvinylpyrrolidone 1g/100ml; 1M and pH8 .0 Tris-HCl buffer solution 8ml/100ml; the solvent is water.

Buffer B is composed of solute and solvent: solute and its final concentration in buffer B are as follows: 1M Tris-HCl buffer solution with pH=8.0 8ml/100ml, disodium edetate 20mmol·L ^-1 , NaCl 1.2M, CTAB 2.5g/100ml, sodium metabisulfite 0.5g/100ml, sodium ascorbate 0.5g/100ml, polyvinylpyrrolidone 1g/100ml, β-mercaptoethanol 80ul/100ml; the solvent is water.

The time of the ice bath was 10 minutes; the condition of the water bath was 90 minutes at 60°C.

Embodiment 3,

The method is basically the same as in Example 1, the differences are as follows:

Buffer A is composed of solute and solvent; the solute and its final concentration in buffer A are as follows: disodium edetate 6mmol·L ^-1 , NaCl 0.3M, polyvinylpyrrolidone 3g/100ml; 1M and pH8 .0 Tris-HCl buffer solution 12ml/100ml; the solvent is water.

Buffer B is composed of solute and solvent: solute and its final concentration in buffer B are as follows: 1M Tris-HCl buffer solution with pH=8.0 12ml/100ml, disodium edetate 30mmol·L ^-1 , NaCl 1.6M, CTAB 3.5g/100ml, sodium metabisulfite 1.5g/100ml, sodium ascorbate 1.5g/100ml, polyvinylpyrrolidone 3g/100ml, β-mercaptoethanol 120ul/100ml; the solvent is water.

The time of the ice bath was 20 minutes; the condition of the water bath was 120 minutes at 70°C.

Comparative example 1,

The plant DNA extraction kit sold by Company I on the market was used to extract according to the instructions. Hereinafter referred to as Company I.

Comparative example 2,

The plant DNA extraction kit sold by Company II on the market was used to extract according to the instructions. Hereinafter referred to as Company II.

Comparative example 3,

The plant DNA extraction kit sold by Company III on the market was used to extract according to the instructions. Hereinafter referred to as Company III.

Comparative example 4,

The plant DNA extraction kit sold by Company IV on the market was used to extract according to the instructions. Hereinafter referred to as Company IV.

The result is as follows:

1. DNA yield

Electrophoresis results: Different DNA extraction methods have different DNA yields due to different cell lysis methods and different DNA capture degrees in subsequent operations. The DNA yield can be judged from the brightness of the DNA electrophoresis strips. Among the above five methods, the electrophoresis band of the DNA extracted by the mCTAB method of Example 1 is obviously brighter than the DNA electrophoresis band of other methods (Fig. 1), indicating that the yield of the DNA extracted by the mCTAB method of Example 1 is high.

Spectrophotometer measurement result (table 2): in five kinds of methods, the DNA productive rate that the mCTAB method of embodiment 1 extracts is obviously higher than other four kinds of kit methods, and the average value of 10 kinds of plants is higher than the average value of each kit method more than double. Especially for sample No. 5 grapes, the mCTAB method in Example 1 extracted more DNA, and the DNA extraction kit of Company I only extracted a small amount of DNA, while Company II, Company III, and Company IV basically did not obtain DNA , indicating that for difficult materials, the mCTAB method of Example 1 has a good extraction effect, and the effect of the kit of company I is not satisfactory.

Table 2. DNA yields estimated spectrophotometrically for 10 plants (nanogram DNA per mg dry material, ng.mg ⁻¹ )

2. DNA quality

In the prior art, it is generally believed that the A260/280 ratio of high-purity DNA should be between 1.8 and 2.0 when detected by an ultraviolet spectrophotometer. When A260/280 is less than 1.8, it indicates that there is protein contamination in the DNA sample, and when A260/280 is greater than 2.0, it indicates that the RNA content in the DNA sample is high. It can be seen from Table 3 that there are certain differences in the purity of DNA extracted by different methods. Seven samples of the DNA extracted by the mCTAB method in Example 1 had A260/280 ratios between 1.8 and 1.89. The A260/280 ratio of 9 samples of the DNA extracted by the company I kit is greater than 2.0, indicating that the RNA content in the DNA sample is relatively high. The A260/280 ratios of most samples of DNA extracted by the kits of Company II, Company III and Company IV were all less than 1.8, indicating that there was pollution of organic matter such as protein in DNA samples. It can be seen that, the DNA purity extracted by the mCTAB method of Example 1 is the highest.

Table 3. DNA purity (A260/280) of 10 plants extracted by five methods.

samples mCTAB I II III IV 1 1.81 3.06 1.71 1.70 1.73 2 1.89 2.25 1.74 1.52 1.77 3 1.80 2.11 1.80 1.67 1.77 4 1.88 2.09 1.81 1.79 1.73 5 1.66 5.69 1.66 1.82 1.60 6 1.85 4.51 1.61 1.25 1.79 7 2.16 2.14 1.71 1.75 1.79 8 1.68 1.98 1.25 1.29 1.47 9 1.83 2.21 1.78 1.80 1.06 10 1.88 2.12 1.83 1.84 1.84

The success rate of PCR amplification is one of the important basis for judging the quality of DNA. As can be seen from the overall PCR amplification success rate in Figure 2, mCTAB in Example 1 is the same as Company I, with the most amplified bands; Company II is the same as Company IVPCR, with slightly fewer amplified bands; Company III is the worst . In terms of specific gene fragments, mCTAB of Example 1 and matK and ITS of Company I are obviously superior to other methods.

3. Cost accounting

The cost of DNA extraction is one of the factors that must be considered when research funding is limited. After calculation, the cost of the mCTAB method in Example 1 is 0.6 yuan for a single sample, the cost of the kits for companies I and II is 5 yuan for a single sample, the cost for company III is 11 yuan for a single sample, and the cost for company IV is 1 yuan for a single sample It is 30 yuan. Obviously, the mCTAB method has a very significant cost advantage.

The methods shown in the above-mentioned Examples 2 and 3 also extracted the target DNA, and the results were not significantly different from those in Example 1.

In summary, the mCTAB method of the present invention for extracting plant DNA has five advantages of high yield, good quality, high PCR amplification success rate, low cost, and good versatility, and can meet the needs of DNA extraction in general molecular biology research. It is especially suitable for researchers with insufficient scientific research funds and the extraction of large quantities of plant DNA.

Claims (9)

1. A test kit for extracting plant DNA, characterized in that: the test kit includes buffer A; The buffer A is composed of a solute and a solvent; the solute and its final concentration in the buffer A are as follows: disodium edetate 4-6mmol·L ^-1 , NaCl 0.2-0.3M, poly 1-3g/100ml of vinylpyrrolidone; 8-12ml/100ml of Tris-HCl buffer solution with a concentration of 1M and a pH of 8.0; the solvent is water. 2. The kit according to claim 1, characterized in that: buffer B is also included in the kit; the buffer B is made up of solute and solvent: the solute and its The final concentrations are as follows: Tris-HCl buffer solution with a concentration of 1M and a pH of 8.0 8-12ml/100ml, disodium edetate 20-30mmol·L ^-1 , NaCl 1.2-1.6M, CTAB 2.5-3.5g /100ml, sodium metabisulfite 0.5-1.5g/100ml, sodium ascorbate 0.5-1.5g/100ml, polyvinylpyrrolidone 1-3g/100ml, β-mercaptoethanol 80-120ul/100ml; the solvent is water. 3. The test kit according to claim 1 or 2, characterized in that: in the buffer A, the solute and its final concentration in the buffer A are as follows: EDTA disodium 5mmol ·L ^-1 , NaCl 0.25M, polyvinylpyrrolidone 2g/100ml; Tris-HCl buffer solution with a concentration of 1M and a pH of 8.0 10ml/100ml; In the buffer B, the solute and its final concentration in the buffer B are as follows: Tris-HCl buffer solution 10ml/100ml with a concentration of 1M and a pH of 8.0, disodium edetate 25mmol. L ^-1 , NaCl 1.4M, CTAB 3g/100ml, sodium metabisulfite 1g/100ml, sodium ascorbate 1g/100ml, polyvinylpyrrolidone 2g/100ml, β-mercaptoethanol 100ul/100ml. 4. A method for extracting plant DNA, comprising the steps of: (1) Mix the buffer solution A with the plant tissue powder, ice bath, centrifuge, discard the supernatant, and collect the precipitate; (2) Mix buffer B with the precipitate obtained in step (1), place, centrifuge, and collect the supernatant; (3) DNA is extracted from the supernatant obtained in step (2) with chloroform isoamyl alcohol solution, and then the steps of removing RNA and precipitating DNA are carried out to obtain the target DNA; The buffer A and the buffer B are both the buffer A and the buffer B in the kit according to any one of claims 1-3. 5. The method according to claim 4, characterized in that: in the step (1), the time of the ice bath is 10min-20min. 6. The method according to claim 4 or 5, characterized in that: in the step (2), the placing method is a 60°C-70°C water bath for 90-120min, specifically a 65°C water bath for 100min. 7. The method according to any one of claims 4-6, characterized in that: in the method, after the step (1) and before the step (2), the following repeating steps are also included: The precipitate obtained in the first step is mixed with the buffer A, ice-bathed, centrifuged, the supernatant is discarded, and the precipitate is collected; until the supernatant is not viscous. 8. The method according to any one of claims 4-7, characterized in that: In the step (1), the ratio of the buffer A to the plant tissue powder is 1mL buffer A: 20mg plant tissue powder; invert and mix 2-3 times during the ice bath; the centrifugation is 7000 × g centrifugal 10min; the time of the ice bath is 15min; In the step (2), the ratio of the buffer B to the plant tissue powder is 0.7mL buffer B: 20mg plant tissue powder; the water bath process is inverted and mixed several times; the centrifugation is 10000 Centrifuge at × g for 10 min. In described step (3), the method for extracting DNA from the supernatant obtained in step (2) with chloroform isoamyl alcohol solution is as follows: add 0.7mL chloroform isoamyl alcohol to step (2) gained supernatant solution, mixed upside down for 10min, 10000×g, centrifuged for 10min, and collected the supernatant; the volume ratio of chloroform:isoamyl alcohol in the chloroform isoamyl alcohol solution was 24:1; the above-mentioned chloroform isoamyl alcohol step was repeated until two There is no precipitation between the liquid surfaces; then add 0.5mL isopropanol, mix well, place at -20°C for 20min, centrifuge at 10000×g for 10min, and discard the supernatant; In the step (3), the method for removing RNA includes the following steps: adding 0.1 mL of RNase solution with a concentration of 100 mg L-1, placing it at 37° C. for 30-60 min; adding 0.1 mL of deionized water, 0.1 mL of 5M NaCl Solution, 0.8mL95% absolute ethanol, mix well, centrifuge at 10000×g for 10min, discard the supernatant; In the step (3), the method for precipitating DNA is as follows: add 0.5mL 75% aqueous ethanol, bounce the precipitate, centrifuge at 10000 × g for 2min, and discard the supernatant. 9. The method according to any one of claims 4-8, wherein the plant tissue is a plant leaf.

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