CN102140451A - Method for extracting DNA (Desoxyribonucleic Acid) and RNA (Ribonucleic Acid) - Google Patents

Abstract

The invention relates to a method for extracting DNA (Desoxyribonucleic Acid) and RNA (Ribonucleic Acid) from prokaryotic or eukaryotic materials, comprising the following steps: (1) pretreatment: cracking prokaryotic or eukaryotic materials by liquid nitrogen grinding method, or by repeated blowing-sucking method with liquid-moving machine suction head, or by glass bead method, and then carrying out dissolving treatment by using decontaminating reagents to obtain a cracked digestion product; (2) adding saturated saline solution to the cracked digestion product and then carrying out protein precipitation for the biological materials; and (3) separating precipitation phase from liquid phase, and then separating and purifying the DNA or RNA in the liquid phase. Compared with prior art, the invention has the advantages that (1) non-toxicity reagent is used; (2) the extraction method is convenient and rapid; (3) the obtained DNA or RNA has high quality and is completely suitable for downstream molecular biology experiments; and (4) the cost is low.

Description

A method for extracting DNA and RNA

technical field

The present invention relates to biotechnology, in particular to a method for extracting DNA and RNA.

Background technique

With the rapid development of biotechnology, the extraction technology of DNA and RNA is becoming increasingly mature and developed. At present, a large number of technologies have been widely used in laboratories, industrial production, pharmaceuticals, animal and plant quarantine, and a large number of related patents have also been produced. Such as "a magnetic nanoparticle complex for nucleic acid separation and its preparation and application (200610023144.4)" and "method for isolating nucleic acid" (200680034578.2), etc.

DNA and RNA are always bound together with various proteins in cells. The separation of DNA and RNA mainly refers to the separation of DNA and RNA from biological macromolecules such as proteins, polysaccharides, and fats. When separating DNA and RNA, the integrity of the primary structure of DNA and RNA molecules should be ensured, and other molecular contamination should be excluded. However, because DNA and RNA extraction samples contain carbohydrates, tannic acid, polyphenols and proteins, and even organic reagents such as phenol and chloroform used in extraction will contaminate the samples, how to obtain high-purity DNA and RNA has become all problems that researchers have to face.

The object of the present invention is to provide a method for extracting DNA and RNA.

Contents of the invention

The object of the present invention is to provide a method for extracting nucleic acid, ie DNA or RNA, from prokaryotic or eukaryotic source material.

The present invention is developed by utilizing the negatively charged characteristics of the phosphate groups of nucleotide molecules. The positive charges in saturated saline are used to neutralize the negative charges of the above phosphate groups, followed by precipitation and separation, and finally purified to obtain DNA or RNA .

Specifically, the method for extracting DNA or RNA of the present invention comprises the following steps:

1) Pretreatment: lysing prokaryotic or eukaryotic biological materials and dissolving them with a detergent to obtain cleavage and digestion products;

2) adding saturated saline to the lysed digestion product, and precipitating the protein in the biological material;

3) Separating the precipitation phase and the liquid phase, and then separating and purifying the DNA or RNA in the liquid phase.

Wherein, the brine described in step 2) is neutral or acidic saturated brine, if it is necessary to extract DNA, then use neutral saturated brine; if it is necessary to extract RNA, then use acidic saturated brine with a pH value of 5-6;

Wherein, the volume dosage of the saturated saline is 0.3-1 times of the volume of the cracked digestion product;

The neutral saturated brine is prepared by directly dissolving NaCl in pure water and boiling; the acid saturated brine is obtained by adding hydrochloric acid to the neutral saturated brine to make the pH value reach 5-6.

Step 2) is carried out as follows: first add 350 μl of neutral saturated salt solution to each ml of the lysed digestion product; then gently invert the above solution to mix, and then place it at room temperature for 5-15 minutes;

Step 3) can be carried out according to techniques known in the art, for example, it can be carried out as follows: the above solution is centrifuged at room temperature 2500rpm for 15-30min to obtain a supernatant containing DNA or RNA, and then the following method is used for the supernatant containing DNA:

1. Precipitate DNA

1.1 Transfer the DNA-containing supernatant to a new Eppendorf tube;

1.2 Centrifuge at 12000rpm for 10min at room temperature, discard the supernatant;

2. Wash DNA

2.1 Add 75% ethanol to DNA precipitation;

2.2 Centrifuge at 10,000 rpm for 5 minutes at room temperature, discard the supernatant;

3. Dissolving DNA

3.1 Dry DNA precipitation at room temperature for 5 minutes, then dissolve in ultrapure water;

3.2 Quantify the obtained DNA and store in -80°C.

4. Removal of RNA Contaminants

4.1 Add 50 μg of RNase to each ml of DNA solution;

4.2 Incubate at 37°C for 1 hour.

NOTE: The RNase treatment used to remove RNA contamination should only be used in the final step of the extraction. If desalting is required to obtain high-quality DNA, steps 3 and 4 of this method can be used after RNase treatment. To preserve DNA for a long time, Tris-EDTA (TE) solution can be used to dissolve DNA to inhibit nuclease activity.

For RNA-containing supernatants use the following method:

1. RNA precipitation

1.1 Transfer the RNA-containing supernatant to a new Eppendorf tube;

1.2 Centrifuge at 12000rpm for 10min at room temperature, discard the supernatant;

2. RNA washing

2.1 Add 75% ethanol to RNA precipitation;

2.2 Centrifuge at 10,000 rpm for 5 minutes at room temperature, discard the supernatant;

3. RNA Dissolution

3.1 Dry the RNA pellet at room temperature for 5 minutes, and then dissolve it in DEPC-treated ultrapure water;

3.2 Quantify the obtained RNA and store in -80°C.

4. Removal of Genomic DNA Contaminants

This part can be operated with DNase I (RNase-free) kit, and the specific steps can be found in the manufacturer's instructions.

In step 1), according to the source and quantity of biological material, the pretreatment can be carried out as follows:

1) For a relatively large amount of biological material, liquid nitrogen grinding method and subsequent dissolution treatment with detergent are used;

2) For a single animal cell or bacterial culture, use a pipette tip to repeatedly blow and suck and then dissolve it with a detergent;

3) For other biological materials including parasite eggs, plants, etc., the glass bead disruption method followed by dissolution treatment with detergent can be used.

The following is a detailed description of the preprocessing method:

1) Liquid nitrogen grinding method to extract DNA or RNA:

1.1 Take the tissue to be tested, chop it and place it in a mortar;

1.2 Add liquid nitrogen to freeze the tissue;

1.3 Grind the tissue with a pestle;

1.4 Add 3ml of lysate to each g of tissue to be tested;

1.5 Add 300 μl of 10% SDS per g of tissue to be tested;

1.6 Transfer the grind to an Eppendorf tube;

1.7 Add proteinase K and digest at 50°C for 1 hour;

2) Pipette repeated blowing method to extract DNA or RNA (take the nucleic acid extraction of Escherichia coli as an example)

1.1 Transfer the bacterial culture or cell culture to an Eppendorf tube, centrifuge at 2000rpm for 3-10min to obtain the precipitate

1.2 Discard the supernatant, add the lysate to the pellet and inhale 8-10 times with a pipette tip to lyse the bacteria or cells;

1.3 Add 100 μl 10% SDS to each milliliter of bacterial culture;

1.4 Add 100 μg of proteinase K to each ml of lysate and incubate in a water bath at 50°C for 1 hour.

3) Extraction of DNA or RNA by glass bead crushing method

1.1 Mix the biological material with sterilized glass beads and shake for 10 minutes to break;

1.2 Add 300μl 10% SDS per ml;

1.3 Add 300 μg proteinase K to each ml of lysate, then incubate at 50°C for 1h°C.

Wherein, the sterilized glass beads are glass beads commonly used in the field, preferably with a diameter of 0.4 mm. The shaking is carried out by a high-speed vortex oscillator with a rotation speed of 5000 rpm.

The core of the invention is to neutralize the negative charge of nucleic acid with cations in salt solution, and the neutralized DNA or RNA is precipitated in high-salt solution. Most impurities such as cell debris and protein can be removed by low-speed centrifugation, and DNA or RNA can be precipitated by ethanol precipitation. Compared with the prior art, the present invention has the advantages of: 1) use of non-toxic reagents; 2) simple and quick extraction method; 3) low cost.

Description of drawings

Figure 1. Genomic DNA (first and second lanes) of Eimeria praecox extracted using the method of the present invention. The DNA molecular weight marker (M) is the λ genome digested by EcoT14.

Figure 2. Genomic DNA of Eimeria tenella extracted using the method of the present invention. The DNA molecular weight marker is the λ genome digested with EcoT14.

Figure 3. Total RNA of Eimeria praecox extracted using the method of the present invention. The DNA molecular weight marker is Trans DNA Marker III.

Figure 4. Total RNA of Eimeria tenella (Eimeria tenella) Howden strain (Hougton Strain) extracted using the method of the present invention. The DNA molecular weight marker is Trans DNA Marker III.

Fig. 5 is the MIC2 gene successfully amplified from the genomes of Eimeria tenella (Eimeria tenella) Howden strain (Hougton Strain) and Beijing strain (Beijing Strain) extracted by the method of the present invention.

Figure 6. Genomic DNA of BALB/c mice extracted using the method of the present invention. The DNA molecular weight marker (M) is the λ genome digested by EcoT14.

FIG. 7 is the 16S ribosomal DNA gene obtained by successfully amplifying the mouse genome extracted using the method of the present invention.

Figure 8. Genomic DNA of Escherichia coli extracted using the method of the present invention. The DNA molecular weight marker (M) is the λ genome digested by EcoT14.

Figure 9 shows the total RNA of Escherichia coli extracted using the method of the present invention. The DNA molecular weight marker is Trans DNA Marker III.

FIG. 10 . The 16S ribosomal DNA gene obtained after the genome of Escherichia coli extracted by the method of the present invention is successfully amplified.

Detailed ways

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

Example 1

The DNA or RNA of the Eimeria oocyst sample was extracted by the glass bead crushing method, and the operation steps were as follows:

1. DNA extraction operation scheme

1. Cell disruption:

1.1 5×10 ⁶ sporulated or unsporulated oocyst pellets were mixed with 0.5 g of sterilized glass beads (0.4 mm in diameter), put into a 15 ml centrifuge tube with a pointed bottom screw cap, and vortexed with a high-speed shaker (5000 rpm ) shake for 10 minutes to break up the oocysts;

1.2 Add 300μl 10% SDS per ml;

1.3 Add 300 μg proteinase K, and then incubate at 50°C for 1 hour.

2. Phase separation:

2.1 Add 1ml of neutral saturated salt solution NaCl to the above lysate;

2.2 Gently invert the centrifuge tube and mix for 15 seconds, then place it at room temperature for 10 minutes;

2.3 Centrifuge at 2500rpm for 15min at room temperature;

2.4 DNA molecules only exist in the supernatant aqueous phase at this time (see the figure below)

3. DNA precipitation

3.1 Transfer the DNA-containing supernatant to a new Eppendorf tube;

3.2 Centrifuge at 12000rpm for 10min at room temperature, discard the supernatant

1.3. DNA precipitation

4. DNA washing

4.1 Add 75% ethanol to DNA precipitation;

4.2 Centrifuge at 10,000 rpm for 5 minutes at room temperature, discard the supernatant;

5. DNA dissolution

5.1 Dry DNA precipitation at room temperature for 5 minutes, then dissolve in ultrapure water;

5.2 Quantify the obtained DNA and store in -80°C.

6. Removal of RNA Contaminants

6.1 Add 50 μg of RNase to each ml of DNA solution;

6.2 Incubate at 37°C for 1 hour.

NOTE: The RNase treatment used to remove RNA contamination should only be used in the final step of the extraction. If desalting is required to obtain high-quality DNA, steps 3 and 4 of this method can be used after RNase treatment. To preserve DNA for a long time, Tris-EDTA (TE) solution can be used to dissolve DNA to inhibit nuclease activity.

2. RNA extraction operation scheme

1. Cell Lysis

1.1 5×10 ⁶ sporulated or unsporulated oocysts were mixed with 0.5 g of sterilized glass beads (0.4 mm in diameter), put into a 15 ml centrifuge tube with a pointed bottom screw cap, and vortexed with a high-speed shaker (5000 rpm) ) shake for 10 minutes to break up the oocysts;

1.2 Add 300 μl 10% SDS to each ml of the above product and mix well, then incubate at 42°C for 1h.

2. Phase separation:

2.1 Add 1 ml of acidic saturated salt solution NaCl with a pH of 5.6 to the above lysate;

2.2 Gently invert the centrifuge tube and mix for 15 seconds, then place it at room temperature for 10 minutes;

2.3 Centrifuge at 2500rpm for 15min at room temperature;

2.4 RNA molecules only exist in the supernatant aqueous phase at this time (see the figure below)

3. RNA precipitation

3.1 Transfer the RNA-containing supernatant to a new Eppendorf tube;

3.2 Centrifuge at 12000 rpm for 10 min at room temperature, discard the supernatant;

4. RNA washing

4.1 Add 75% ethanol to RNA precipitation;

4.2 Centrifuge at 10,000 rpm for 5 minutes at room temperature, discard the supernatant;

5. RNA Dissolution

5.1 Dry the RNA pellet at room temperature for 5 minutes, and then dissolve it in DEPC-treated ultrapure water;

5.2 Quantify the obtained RNA and store in -80°C.

6. Then according to the instructions, use DNase I (RNase-free) to remove genomic DNA pollutants

3. DNA extraction results

The ratio of the extracted DNA A26o/A28o was 1.87, and its quality fully met the requirements of subsequent molecular biology operations. The yield of DNA depends on the number of initial oocysts, that is, 3.5985 mg DNA can be obtained from 6×10 ⁶ sporulated oocysts. Figure 1 and Figure 2 are 1.5% agarose gel electrophoresis images of the extracted DNA.

4. Results of RNA extraction

The ratio of extracted RNA A26o/A28o was 1.42, and its quality fully met the requirements of subsequent molecular biology operations. The yield of DNA depends on the number of starting oocysts, that is, 191.7 μg RNA can be obtained from 6×10 ⁶ sporulated oocysts. Figure 3 and Figure 4 are 1.5% agarose gel electrophoresis images of the extracted RNA.

5. Application of extracted genomic DNA and RNA in downstream operations

Specific primers can be used to PCR amplify specific target genes from the extracted DNA or RNA to verify the applicability of the extracted DNA or RNA. In this example, MIC2-UP (tatggctcgagcgttgtcgctg) and MIC2-D (gtcaggatgactgttgagtgtc) were used to amplify the MIC2 gene (GENBANK accession number FJ807654) from the extracted Eimeria tenella genomic DNA. The primers used were synthesized by Beijing Aoke Biological Co., Ltd.

Fig. 5 shows the MIC2 gene amplified from the genomic DNA of Eimeria tenella Beijing strain and Howden strain.

Example 2

Use the liquid nitrogen grinding method to extract DNA or RNA from mouse cells. The operation steps are as follows:

A technical solution for extracting DNA by liquid nitrogen grinding

1. Grinding

1.1 Take 1g of tissue, chop it and place it in a mortar;

1.2 Add liquid nitrogen to freeze the tissue;

1.3 Grind the tissue with a pestle;

1.4 Add 3ml of lysate;

1.5 Add 300 μl of 10% SDS;

1.6 Transfer the grind to an Eppendorf tube;

1.7 Add proteinase K for digestion (50℃℃1h)

2. Extraction

2.1 Add 350 μl neutral saturated salt solution (NaCl) to each ml of the lysed digestion product

2.2 Gently invert and mix the above solution for 15 seconds, then place it at room temperature for 10 minutes;

2.3 Centrifuge at 2500 rpm for 15 minutes at room temperature, and the supernatant aqueous phase contains DNA.

3. DNA precipitation

3.1 Transfer the DNA-containing supernatant to a new Eppendorf tube;

3.2 Centrifuge at 12000 rpm for 10 min at room temperature, discard the supernatant;

4. DNA washing

4.1 Add 75% ethanol to DNA precipitation;

4.2 Centrifuge at 10,000 rpm for 5 minutes at room temperature, discard the supernatant;

5. DNA dissolution

5.1 Dry DNA precipitation at room temperature for 5 minutes, then dissolve in ultrapure water;

5.2 Quantify the obtained DNA and store in -80°C.

6. Removal of RNA Contaminants

6.1 Add 50 μg of RNase to each ml of DNA solution;

6.2 Incubate at 37°C for 1 hour.

NOTE: The RNase treatment used to remove RNA contamination should only be used in the final step of the extraction. If desalting is required to obtain high-quality DNA, steps 3 and 4 of this method can be used after RNase treatment. To preserve DNA for a long time, Tris-EDTA (TE) solution can be used to dissolve DNA to inhibit nuclease activity.

2. Technical scheme of RNA extraction by liquid nitrogen grinding method

1. Grinding

1.1 Take 1g of tissue, chop it and place it in a mortar;

1.2 Add liquid nitrogen to freeze the tissue;

1.3 Grind the tissue with a pestle;

1.4 Add 3ml of lysate;

1.5 Add 300 μl of 10% SDS;

1.6 Transfer the grind to an Eppendorf tube;

1.7 Add proteinase K for digestion (50°C 1h)

2. Extraction

2.1 Add 350 μl of acidic saturated salt solution (NaCl) at pH 6 to each ml of the lysed digest

2.2 Gently invert and mix the above solution for 15 seconds, then place it at room temperature for 10 minutes;

2.3 Centrifuge at 2500 rpm for 15 minutes at room temperature, and the supernatant aqueous phase contains RNA.

3. RNA precipitation

3.1 Transfer the RNA-containing supernatant to a new Eppendorf tube;

3.2 Centrifuge at 12000 rpm for 10 min at room temperature, discard the supernatant;

4. RNA washing

4.1 Add 75% ethanol to RNA precipitation;

4.2 Centrifuge at 10,000 rpm for 5 minutes at room temperature, discard the supernatant;

5. RNA Dissolution

5.1 Dry the RNA pellet at room temperature for 5 minutes, and then dissolve it in DEPC-treated ultrapure water;

5.2 Quantify the obtained RNA and store in -80°C.

6. Removal of Genomic DNA Contaminants

This part can be operated with DNase I (RNase-free) kit, and the specific steps can be found in the manufacturer's instructions.

3. DNA extraction effect

The ratio of A26o/A28o of the extracted DNA was 1.83, and its quality fully met the requirements of subsequent molecular biology operations. The yield of DNA depends on the amount of starting tissue material, ie 200 μg of DNA can be obtained from 10 mg of tissue. Fig. 6 is a 1.5% agarose gel electrophoresis image of the extracted DNA.

Use of the genomic DNA and RNA extracted by the present invention in downstream operations

Specific primers can be used to PCR amplify specific target genes from the extracted DNA or RNA to verify the applicability of the extracted DNA or RNA. In this example, forward GAPDH Primer (5'-CAAGGTCATCCATGACAACTTTG-3' and reverse GAPDH Primer (5'-GTCCACCACCCCTGTTGCTGTAG-3') were used to amplify 16S ribosomal DNA from the extracted mouse genomic DNA. The used The primers were synthesized by Beijing Aoke Biological Co., Ltd. The results are shown in Figure 7.

Example 3

Use the pipette tip to repeatedly blow and suck to extract the DNA or RNA of Escherichia coli. The operation steps are as follows:

1. DNA extraction operation scheme

1 homogenate

1.1 Transfer 1ml of the bacterial culture to an Eppendorf tube and centrifuge at 2000rpm for 5min to obtain a precipitate

1.2 Discard the supernatant, add 1ml of lysate to the pellet, and blow and suck the lysed bacteria repeatedly with the pipette tip

1.3 Add 100μl 10% SDS;

1.4 Add 100 μg of proteinase K and incubate in a water bath at 50°C for 1 hour.

2. Water phase separation:

2.1 Add 350 μl neutral saturated salt solution (NaCl) to each ml of the lysed digestion product;

2.2 Gently invert and mix the above solution for 15 seconds, then place it at room temperature for 10 minutes;

2.3 Centrifuge at 2500 rpm for 15 minutes at room temperature, and the supernatant aqueous phase contains DNA.

3. DNA precipitation

3.1 Transfer the DNA-containing supernatant to a new Eppendorf tube;

3.2 Centrifuge at 12000rpm for 10min at room temperature, discard the supernatant

1.3. DNA precipitation

4. DNA washing

4.1 Add 75% ethanol to DNA precipitation;

4.2 Centrifuge at 10,000 rpm for 5 minutes at room temperature, discard the supernatant;

5. DNA dissolution

5.1 Dry DNA precipitation at room temperature for 5 minutes, then dissolve in ultrapure water;

5.2 Quantify the obtained DNA and store in -80°C.

6. Removal of RNA Contaminants

6.1 Add 50 μg of RNase to each ml of DNA solution;

6.2 Incubate at 37°C for 1 hour.

NOTE: The RNase treatment used to remove RNA contamination should only be used in the final step of the extraction. If desalting is required to obtain high-quality DNA, steps 3 and 4 of this method can be used after RNase treatment. To preserve DNA for a long time, Tris-EDTA (TE) solution can be used to dissolve DNA to inhibit nuclease activity.

2. RNA extraction operation scheme

1. Homogenization

1.1 Transfer 1ml of the bacterial culture to an Eppendorf tube and centrifuge at 2000rpm for 5min to obtain a precipitate

1.2 Discard the supernatant, add 1ml of lysate to the pellet, and blow and suck the lysed bacteria repeatedly with the pipette tip

1.3 Add 100 μl of 10% SDS;

1.4 Add 100 μg of proteinase K and incubate in a water bath at 50°C for 1 hour.

2. Phase separation

2.1 Add 350 μl of acidic saturated salt solution (NaCl) at pH 5 to each ml of the lysed digest

2.2 Gently invert and mix the above solution for 15 seconds, then place it at room temperature for 10 minutes;

2.3 Centrifuge at 2500 rpm for 15 minutes at room temperature, and the supernatant aqueous phase contains RNA.

3. RNA precipitation

3.1 Transfer the RNA-containing supernatant to a new Eppendorf tube;

3.2 Centrifuge at 12000 rpm for 10 min at room temperature, discard the supernatant;

4. RNA washing

4.1 Add 75% ethanol to RNA precipitation;

4.2 Centrifuge at 10,000 rpm for 5 minutes at room temperature, discard the supernatant;

5. RNA Dissolution

5.1 Dry the RNA pellet at room temperature for 5 minutes, and then dissolve it in DEPC-treated ultrapure water;

5.2 Quantify the obtained RNA and store in -80°C.

6. Removal of Genomic DNA Contaminants

This part can be operated with DNase I (RNase-free) kit, and the specific steps can be found in the manufacturer's instructions.

3. DNA separation effect:

The ratio of A26o/A28o of the extracted DNA was 1.83, and its quality fully met the requirements of subsequent molecular biology operations. The yield of DNA depends on the amount of starting tissue material, that is, 48 μg of DNA can be finally obtained from 1 ml of overnight cultured E. coli. Fig. 8 is a 1.5% agarose gel electrophoresis image of the extracted DNA.

4. RNA Extraction

The ratio of extracted RNA A26o/A28o was 1.99, and its quality fully met the requirements of subsequent molecular biology operations. The yield of RNA depends on the number of starting oocysts, that is, 22 μg RNA can be obtained from 1×10 ⁶ E. coli. Fig. 9 is a 1.5% agarose gel electrophoresis image of extracted RNA.

5. Use the genomic DNA and RNA extracted by the present invention for downstream operations

Specific primers can be used to PCR amplify specific target genes from the extracted DNA or RNA to verify the applicability of the extracted DNA or RNA. In this example, multiple 16S ribosomal DNA genes (gene accession numbers J01859/K02555/M24828/M24833/M24834/M24835/M24836/M24837/M24911/M24996) against E. Amplification of 16S ribosomal DNA. The primers used were synthesized by Beijing Aoke Biological Co., Ltd. The results are shown in Figure 10.

Claims (6)

1. a method of extracting DNA or RNA from protokaryon or eukaryote source material is characterized in that, comprises the steps:

1) pre-treatment: cracking protokaryon or eukaryote material also carry out dissolution process with stain remover, obtain the cracking digestion product;

2) saturated aqueous common salt is joined in the cracking digestion product, the albumen in the biomaterial is carried out precipitation process;

3) precipitation separation mutually and liquid phase, then to DNA in the liquid phase or RNA separates and purifying.

2. the method for claim 1 is characterized in that step 2) described salt solution is neutral or acid saturated brine, if need obtain DNA, then uses neutral saturated brine; If need obtain RNA, then use the acid saturated brine of pH value as 5-6.

3. method as claimed in claim 1 or 2 is characterized in that step 2) volumetric usage of described saturated aqueous common salt be cracking digestion product volume 0.3-1 doubly.

4. the method for claim 1 is characterized in that, in step 1), described cracking adopts the liquid nitrogen grinding method to carry out.

5. the method for claim 1 is characterized in that, in step 1), described cracking employing pipettor suction nozzle pressure-vaccum method repeatedly carries out.

6. the method for claim 1 is characterized in that, in step 1), described cracking adopts the granulated glass sphere method of breaking to carry out.

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