CN111500571A

CN111500571A - Eukaryotic DNA extraction method suitable for Hi-C technology, rinsing liquid and application

Info

Publication number: CN111500571A
Application number: CN202010271078.2A
Authority: CN
Inventors: 郑洪坤; 康靖民
Original assignee: Qingdao Baimaike Biotechnology Co ltd
Current assignee: Qingdao Baimaike Biotechnology Co ltd
Priority date: 2020-04-08
Filing date: 2020-04-08
Publication date: 2020-08-07

Abstract

The invention relates to the technical field of molecular biology, and particularly discloses a eukaryotic DNA extraction method, rinsing liquid and application suitable for a Hi-C technology.

Description

Eukaryotic DNA extraction method suitable for Hi-C technology, rinsing liquid and application

Technical Field

The invention relates to the technical field of molecular biology, in particular to a method for extracting eukaryotic DNA (deoxyribonucleic acid) suitable for a Hi-C technology, rinsing liquid and application.

Background

With the development of high-throughput sequencing technology, sequencing cost is lower and the scale is larger, and genomes of many species are sequenced. Although long-read sequencing technologies such as Pacbio and Oxford Nanopore exist, genome highly repetitive genome of complex species cannot be assembled to chromosome level, and although genome sequences with different assembly integrity meet most genome research and breeding work, high-quality and even complete reference genomes can be used for more accurate and deep prediction of gene structure and function and excavation of all gene regulatory sequences. Hi-C can correctly splice the Scaffolds belonging to the same chromosome to achieve the chromosome level by the characteristic that the interaction frequency of the chromatin fragments of the same chromosome is high and the interaction frequency of the chromatin fragment sequences of different chromosomes is low.

However, some eukaryotes, especially plants and algae, contain a large amount of polysaccharides, for example, kelp contains algin, fucoidin, alginate starch, etc., which all affect the enzyme activity used in enzyme digestion, which is a key step for determining the success or failure of the Hi-C experiment, and thus the Hi-C experiment fails. And because of the specificity of Hi-C experiment, conformation in cell nucleus must be ensured before enzyme digestion enzyme-linked reaction is finished, so the commonly used sugar removing method of CTAB and the like is not applicable.

At present, a Hi-C extraction process applicable to polysaccharide and polyphenol viscous eukaryotes does not exist. Therefore, it is necessary to provide a method for extracting DNA from eukaryotes, a rinsing solution, and applications suitable for the Hi-C technique to solve the above problems.

Disclosure of Invention

In order to solve the problems in the prior art, the invention aims to provide a eukaryotic DNA extraction method suitable for a Hi-C technology.

In order to achieve the purpose of the invention, the technical scheme of the invention is as follows:

a eukaryotic DNA extraction method suitable for Hi-C technology comprises rinsing crosslinked and wall-broken sample tissue with rinsing solution, and extracting cell nucleus;

the rinsing liquid comprises glycerol, EDTA, PEG, PVP and β -mercaptoethanol.

Preferably, the eukaryote of the present invention is a polysaccharide polyphenol-rich eukaryote.

More preferably, the polysaccharide polyphenol enriched eukaryote is a plant (non-algal) or an algae.

The invention provides a eukaryotic DNA extraction method suitable for a Hi-C technology, which utilizes a special rinsing buffer solution to rinse tissues before cell nucleus extraction, thereby removing polysaccharide polyphenols in a sample under the condition of not destroying the internal conformation of cell nucleus. By using the method, high-quality cell nucleuses can be extracted from the polysaccharide and polyphenol sample, so that the subsequent enzyme digestion and enzyme ligation effect is not influenced by impurities, and difficult sample experiments which cannot be completed by the conventional chromosome conformation capture technical process can be completed.

The cross-linking of the invention adopts a formaldehyde cross-linking method, and the wall breaking adopts a liquid nitrogen grinding method.

The method further comprises the steps of enzyme digestion, marking, enzyme linking, crosslinking release, protein digestion and purification after the extraction of cell nucleus.

In the invention, the rinsing liquid comprises 100-120mM Tris-HCl, 5-10% (v/v) glycerol, 4-6mM EDTA, 5-10% (w/v) PEG (polyethylene glycol), 0.8-1.2% (w/v) PVP (polyvinylpyrrolidone) and 0.8-1.2% (v/v) β -mercaptoethanol.

Preferably, the rinsing liquid comprises 100mM Tris-HCl, 5-10% of glycerol, 5mM EDTA, 5-10% of PEG, 1% of PVP and 1% of β -mercaptoethanol.

More preferably, the rinsing solution comprises 100mM Tris-HCl, 8% glycerol, 5mM EDTA, 8% PEG, 1% PVP, 1% β -mercaptoethanol.

In the invention, the molecular weight of the PEG is 4000-8000; the molecular weight of the PVP is 10000-40000.

In the rinsing of the invention, the ratio of the sample tissue to be detected to the rinsing liquid is 1g (15-30) ml, preferably 1g (22.5 ml), so as to ensure the rinsing effect.

In the invention, the rinsing times are 2-5 times.

The invention repeatedly carries out rinsing (centrifugation after fully mixing with rinsing liquid) steps, and the supernatant fluid after centrifugation is preferably free from sticky feeling.

In the rinsing of the present invention, the conditions of centrifugal separation are: 2500-; preferably 3000g, 4 ℃ and 5 min. The centrifugal condition can precipitate the cell nucleus without damaging the cell nucleus.

In the invention, after the extraction of cell nucleuses, the rinsing liquid is used for secondary rinsing.

According to the invention, after the cell nucleus is extracted, the cell nucleus is rinsed for the second time, so that polysaccharides and polyphenols in the sample can be more sufficiently removed, and the effect of enzyme-linked enzyme digestion in the subsequent step is further ensured not to be influenced by impurities.

In the secondary rinsing of the invention, the ratio of the tissue of the sample to be detected to the rinsing liquid is 1g to 6 ml, preferably 1g to 5ml, so as to ensure the rinsing effect and avoid centrifugal loss.

In the invention, the number of times of the secondary rinsing is 1-3.

In the present invention, in the secondary rinsing, the centrifugal separation conditions are as follows: 1500-; preferably 1800g, 4 ℃ for 5 min. The centrifugal condition can precipitate the cell nucleus without damaging the cell nucleus.

The DNA extraction method specifically comprises the following steps:

1. cross-linking the tissue of the sample to be detected by formaldehyde;

2. terminating the crosslinking reaction with glycine;

3. grinding the tissue of a sample to be detected by liquid nitrogen;

4. after the rinsing liquid is used for resuspending the powder of the tissue of the sample to be tested, centrifuging;

5. repeating the step 4 until the centrifugal supernatant has no sticky feeling;

6. resuspending the precipitate with NIBuffer, filtering to remove impurities, and centrifuging the filtrate;

7. carrying out resuspension precipitation by using NIBTM, carrying out gradient centrifugation, and then collecting cell nuclei;

8. rinsing cell nucleuses with a rinsing solution and then centrifuging;

9. resuspending the pellet with NEBuffer2 and then centrifuging;

10. resuspending the precipitate again with NEBuffer2, and adding SDS for reaction;

11. adding polyethylene glycol octyl phenyl ether for reaction;

12. enzyme digestion;

13. marking;

14. enzyme-linked;

15. de-crosslinking;

16. protein digestion;

17. and (5) DNA purification.

Every 50ml of the NIBuffer comprises the following components: 15-25ml of glycerol, 20XPBS2.5ml, 1M MgCl₂40-60ul, 300ul of 1M KCl 200-.

Preferably, every 50ml of NIBuffer comprises: glycerol 20ml, 20XPBS2.5ml, 1M MgCl₂50ul, 1MKCl 250ul, Roche protease inhibitor 1 tablet, sucrose 4.275g, 10% polyethylene glycol octyl phenyl ether 1.25ml, 100mM phenylmethylsulfonyl fluoride 50ul, β -mercaptoethanol 50 ul.

The NIBTM comprises 8-12mM Tris-HC L, 8-12mM EDTA, 70-90mM KCl, 0.4-0.6M sucrose, 0.8-1.2mM spermine tetra-hydrochloric acid, 0.8-1.2mM spermidine tri-hydrochloric acid, 7-9% polyvinylpyrrolidone, 0.4-0.6% polyethylene glycol octyl phenyl ether, and 7-8% 2-mercaptoethanol.

Preferably, the NIBTM comprises 10mM Tris-HC L, 10mM EDTA, 80mM KCl, 0.5M sucrose, 1mM spermine tetrahydrochloride, 1mM spermidine trihydrochloride, 8% polyvinylpyrrolidone, 0.5% polyethylene glycol octylphenyl ether, 7.5% 2-mercaptoethanol.

As a preferred embodiment, a specific DNA extraction method of the present invention is:

1.2 g of a fresh tender tissue sample is taken and washed clean by ice water;

2. cutting the sample tissue into pieces by using scissors, and placing the pieces into a 50ml centrifuge tube;

3. adding NIBuffer (nucleic acid isolation buffer) 30-50 ml;

4. adding 2ml of 36% formaldehyde solution, and reacting in a rotary hybridization furnace for 90min at room temperature;

5. 2.5ml of 2M glycine is added, and the crosslinking reaction is stopped by continuously shaking for 5 min;

6. filtering off the liquid, and washing with sterilized water until no foam exists;

7. after the moisture on the surface of the tissue is sucked dry, the tissue is placed in liquid nitrogen for full grinding;

8. placing the ground tissue powder into a pre-cooled 50ml centrifuge tube, and placing the centrifuge tube into liquid nitrogen;

9. taking the centrifuge tube filled with the tissue powder out of liquid nitrogen, resuspending the tissue powder with 45ml of rinsing solution (100mM Tris-HCl, 5-10% glycerol, 5mM EDTA, 5-10% PEG, 1% polyvinylpyrrolidone and 1% β -mercaptoethanol), and reversing and mixing evenly for 10 times;

10.3000 g, centrifuging for 5min at 4 ℃, and discarding the supernatant;

11. repeating steps 9 and 10 three times;

12. adding 50ml of NIBuffer to resuspend the precipitate;

13. placing the centrifuge tube with the precipitate on ice, placing on a shaker at 100rpm, and mixing for 15 min;

14. filtering to remove impurities, and collecting filtrate;

15.3000 g, centrifuging at 4 deg.C for 5min, discarding supernatant (if the centrifugation effect is not good, the centrifugation time can be prolonged), and retaining precipitate;

16. adding 4ml of 1 XNIBTM to resuspend the pellet;

17. the resuspended sample was dropped onto the top of a gradient centrifuge (3.75ml PercoII, 1.25ml NIBTM) and centrifuged for 30min at 4 ℃ on a 650g horizontal rotor;

18. sucking out the white liquid layer in the middle of the two layers by using a cut gun head, and transferring the liquid layer into a new 15ml centrifuge tube;

19. adding 10ml rinsing liquid (100mM Tris-HCl, 5-10% glycerol, 5mM EDTA, 5-10% PEG, 1% polyvinylpyrrolidone and 1% β -mercaptoethanol), reversing and mixing evenly;

20.1800 g, centrifuging for 5min at 4 ℃, and discarding the supernatant;

21. adding 500ul 1.2x NEBuffer2 to resuspend and precipitate, transferring to a 1.5ml centrifuge tube, centrifuging at 4 deg.C and 1800g for 5min, discarding supernatant, and retaining precipitate;

22. 500ul of 1.2XNEBuffer 2 was added to resuspend the pellet;

23. adding 7.5ul 20% SDS, 65 deg.C, 900rpm, 20 min;

24. placing on ice for 1 min;

25. adding 50ul of 20% Triton X-100, 37 ℃, 900rpm, 30 min;

26. adding 40ul Hind III or DPN II, performing enzyme digestion at 37 ℃ and 900rpm for 1-16 hours;

27. adding a labeling reaction system (37.5ul of 0.4mM Biotin-14-dCTP, 1.5ul of dA/T/GTP solution and 10ul of Klenow Fragment (5U/U L)) into the enzyme-digested sample, and mixing the mixture well, wherein the mixture is subjected to metal bath at 37 ℃ for 45min (the mixture is mixed in a reverse manner every 5 min), and the dA/T/GTP solution comprises 10mM dATP, 10mM dTTP and 10mM dGTP;

28. taking out the sample tube from the metal bath, and placing the sample tube on ice for 30 s;

29. adding a ligation reaction system (630ul 2x Rapid L ligation Buffer, 6ul T4DNA L igase (DNA ligase)) into the marked sample, fully mixing uniformly, performing metal bath at 20 ℃, and performing 30min (mixing uniformly by reversing every 5 min);

30.5000 rpm, centrifuging for 3min, and discarding the supernatant;

31. adding a crosslinking-releasing system (150ul of nuclease-free water, 20ul of protease K and 10ul of 20% SDS) into the sample tube from which the supernatant is discarded, fully and uniformly mixing, and releasing crosslinking for 2-16 hours at 55 ℃ in a metal bath and 900 rpm;

32. adding 20ul proteinase K into the sample tube, mixing well, and performing metal bath at 65 ℃, 900rpm, 120 min;

33. first purification of DNA:

(1) phenol was added in equal volume to the sample: chloroform: isoamyl alcohol (the volume ratio of the three is 25:24:1) is centrifuged at 8000rpm and 4 ℃;

(2) transferring the supernatant (water phase) into a 50ml centrifuge tube, and supplementing the volume to 15ml with sterile water;

(3) adding 1.5ml 3M sodium acetate, mixing, adding 35ml precooled (-20 deg.C) anhydrous ethanol to make up to 50ml, 20ul glycogen (20ng/ul) or similar substitute;

(4) mixing by inversion, horizontally placing in a refrigerator at-80 deg.C, and incubating for 2 hr or 8-16 hr at-20 deg.C;

(5) centrifuging at 4 deg.C and 10000rpm for 20 min;

(6) discarding the supernatant, washing the DNA precipitate twice with 70% ethanol, 10000rpm, and centrifuging for 5 min;

(7) carrying out air spinning, and removing alcohol completely;

(8) placing the centrifuge tube in an oven for drying;

(9) adding 100ul of nuclease-free water to obtain 100ul of DNA sample;

34. high quality DNA samples were obtained by purification with 120ul magnetic beads.

The invention also provides a eukaryotic DNA extraction rinsing liquid suitable for the Hi-C technology, wherein the rinsing liquid comprises glycerol, EDTA, PEG, PVP and β -mercaptoethanol.

In the invention, the rinsing liquid comprises 100-120mM Tris-HCl, 5-10% (v/v) glycerol, 4-6mM EDTA, 5-10% (w/v) PEG, 0.8-1.2% (w/v) PVP and 0.8-1.2% (v/v) β -mercaptoethanol.

The invention also provides an application of the method or the rinsing liquid in chromosome three-dimensional conformation analysis, gene regulation and control sequence excavation or DNA interaction analysis.

The invention has the beneficial effects that:

the extraction method provided by the invention can remove polysaccharide polyphenol substances in a sample under the condition of not damaging the internal conformation of a cell nucleus, the yield of DNA is high, the enzyme digestion and enzyme ligation effect is good, and the extracted DNA can be suitable for constructing a Hi-C library, so that the Hi-C technology can be applied to the DNA detection of plants and algae rich in polysaccharide polyphenol.

Drawings

FIG. 1 is a schematic diagram of the DNA extraction process of the present invention;

FIG. 2 is an electrophoretogram of DNA of example 1 of the present invention; wherein M1 represents lambda Hind III, M2 represents 15K marker, 1 represents a purified DNA band, 2 represents a DNA band before enzyme digestion, and 3 represents a DNA band after enzyme digestion;

FIG. 3 is a DNA interaction heatmap of example 1 of the present invention;

FIG. 4 is an electrophoretogram of DNA of example 2 of the present invention; wherein M1 represents lambda Hind III, M2 represents 15K marker, 1 represents a purified DNA band, 2 represents a DNA band before enzyme digestion, and 3 represents a DNA band after enzyme digestion;

FIG. 5 is an electrophoretogram of DNA of comparative example 2 of the present invention; wherein M1 represents lambda Hind III, M2 represents 15K marker, 1 represents a purified DNA band, 2 represents a DNA band before cleavage, and 3 represents a DNA band after cleavage.

Detailed Description

Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The main sources of reagents used in the detailed description of the invention are shown in table 1.

TABLE 1

Name of reagent	Manufacturer of the product
		Formaldehyde (I)	Sigma
Glycine	Amresco
		1M Tris-HCl(PH 8.0)	Invitrogen
DPBS powder	Invitrogen
		PVP-40	amresco
β -mercaptoethanol	amresco
		Glycerol	Made in China
PMSF (phenylmethylsulfonyl fluoride)	Biyuntian (a Chinese character)
		Protease inhibitors	Roche of Roche
10×NEBuffer 2	NEB
		SDS	Amresco
Triton X-100	Sigma
		HindIII	NEB
Biotin-14-dCTP	Invitrogen
		100mM dATP	NEB
100mM dGTP	NEB
		100mM dTTP	NEB
Klenow Fragment	NEB
		Nucleic-free water (Nuclease-free water)	Ambion
1M MgCl₂	Sigma
		Potassium chloride	Beijing chemical industry
PEG-8000	Sigma
		T4DNA Ligase	Enzymatics
Proteinase K	Tiangen (root of heaven)
		AMPure XP Beads (magnetic Beads)	Beckman
miracloth	Merck
		PercoII	GE Healthcare
Rapid Ligation Buffer	Enzymatics

Example 1

The present embodiment provides a method for extracting DNA from kelp (see fig. 1 for a schematic flow chart), which includes the following specific extraction steps:

1. taking 2g of fresh kelp, and cleaning with ice water;

2. cutting the thinner leaves at the edge of the kelp into pieces by using scissors, and placing the pieces into a 50ml centrifugal tube;

3. adding 35ml of NIBuffer (nucleic acid isolation buffer);

9. taking the centrifuge tube with the tissue powder out of liquid nitrogen, resuspending the tissue powder with 45ml of rinsing solution (100mM Tris-HCl, 8% glycerol, 5mM EDTA, 8% PEG-8000, 1% PVP-40, 1% β -mercaptoethanol) and reversing and mixing for 10 times;

10.3000 g, centrifuging for 5min at 4 ℃, and discarding the supernatant;

11. repeating steps 9 and 10 three times;

12. adding 50ml of NIBuffer to resuspend the precipitate;

14. filtering with 4 layers of miracloth twice to remove impurities, and collecting filtrate;

16. adding 4ml of 1 XNIBTM to resuspend the pellet;

17. the resuspended sample was dropped onto the upper layer of a gradient centrifugation (3.75ml PercoII, 1.25ml NIBTM, i.e.a concentration of PercoII of 75%) and centrifuged on a 650g horizontal rotor at 4 ℃ for 30 min;

19. adding 10ml rinsing solution (100mM Tris-HCl, 8% glycerol, 5mM EDTA, 8% PEG-8000, 1% PVP-40, 1% β -mercaptoethanol), and mixing;

20.1800 g, centrifuging for 5min at 4 ℃, and discarding the supernatant;

22. 500ul of 1.2XNEBuffer 2 was added to resuspend the pellet;

23. adding 7.5ul 20% SDS, 65 deg.C, 900rpm, 20 min;

24. placing on ice for 1 min;

25. adding 50ul of 20% Triton X-100, carrying out reaction at 37 ℃ and 900rpm for 30min, carrying out heavy suspension precipitation after the reaction is finished, taking out 10ul of heavy suspension, and placing the heavy suspension in a new 1.5ml centrifuge tube to mark the heavy suspension as before enzyme digestion, preserving the heavy suspension at-20 ℃ (subsequently using the heavy suspension for electrophoretic verification before enzyme digestion), and continuing the next experiment on the residual system;

26. adding 40ul Hind III, performing enzyme digestion for 15 hours at 37 ℃, 900rpm, after the reaction is finished, re-suspending the precipitate, taking out 10ul of the re-suspension, marking the re-suspension in a new 1.5ml centrifuge tube as enzyme digestion, storing at-20 ℃ (subsequently used for electrophoresis verification after enzyme digestion), and continuing the next experiment on the rest system;

29. adding a ligation reaction system (630ul 2x Rapid L alignment Buffer, 6ul T4DNA L igase) into the marked sample, fully mixing uniformly, performing metal bath at 20 ℃ for 30min (mixing uniformly by reversing every 5 min);

30.5000 rpm, centrifuging for 3min, and discarding the supernatant;

31. adding a crosslinking-releasing system (150ul of nuclease-free water, 20ul of protease K and 10ul of 20% SDS) into a sample tube from which the supernatant is discarded, fully and uniformly mixing, and releasing crosslinking for 15 hours at 55 ℃ and 900rpm in a metal bath;

33. first purification of DNA:

(3) adding 1.5ml 3M sodium acetate, mixing, adding 35ml precooled (-20 deg.C) anhydrous ethanol to make up to 50ml, 20ul glycogen (20 ng/ul);

(4) the mixture is evenly mixed by inversion, and the mixture is horizontally placed in a refrigerator at the temperature of-80 ℃ for incubation for 2 h;

(5) centrifuging at 4 deg.C and 10000rpm for 20 min;

(7) carrying out air spinning, and removing alcohol completely;

(8) placing the centrifuge tube in an oven for drying;

(9) adding 100ul of nuclease-free water to obtain 100ul of DNA sample;

34. purification with 120ul Beckmann magnetic beads yielded 80ul of high quality DNA samples. 50ng of purified DNA was removed and marked post-purification in a new 1.5ml centrifuge tube (for subsequent post-purification electrophoretic validation).

The NIBuffer formulation used in this example is shown in Table 2.

TABLE 2

The NIBTM formulations used in this example are shown in Table 3.

TABLE 3

This embodiment is further to the kelp DNA who obtains carry out extraction volume detection and electrophoresis experiment, and the electrophoresis experimental result is seen in fig. 2, and the DNA total amount of extracting is 1.6ug, and specific experimentation is:

and (3) detecting the extraction amount of the DNA:

the concentration of 80ul of DNA samples obtained in this example was measured using a QubitTM 4fluorometer (Invitrogen), and the measurement was 20ng/ul, and the volume × concentration was the DNA extraction.

And (3) electrophoresis detection:

1. adding 50ul of 1.2xNEBuffer 2 and 10ul of 20mg/ml protease K into the intermediate products before and after enzyme digestion stored at-20 ℃ in the experiment respectively, and digesting for 30min at 65 ℃;

2. taking 20ul of the digested intermediate product before enzyme digestion, 20ul of the digested intermediate product after enzyme digestion and 50ng of purified DNA, adopting 1% agarose gel and 1 xTAE buffer solution, and carrying out electrophoresis at 70V for 45 min;

3. staining the gel with 1/3000 ethidium bromide diluent (final concentration of ethidium bromide is 3.3ug/ml) for 20 min;

4. the gel imager was used for photographing.

From the above experimental results, it can be seen that the extraction amount of DNA is 1.6ug, which satisfies the Hi-C library building requirement; a complete genome strip exists before enzyme digestion (mark 2 in figure 2), and the extraction method of the invention cannot damage the integrity of the genome; after enzyme digestion (marked with 3 in figure 2), the bands are dispersed, which shows that the method removes impurities which affect the enzyme digestion reaction, so that the enzyme digestion reaction is efficient; after purification (labeled 1 in FIG. 2), a distinct ligation band was observed, indicating that the method of the present invention removes impurities affecting the ligation reaction, making the ligation reaction efficient.

This example further performs Hi-C library construction and sequencing on the obtained DNA, and performs interaction heatmap verification with the sequencing data, the verification result is shown in fig. 3, and the specific process is as follows:

1. ultrasonically breaking the obtained DNA;

2. capturing a target fragment;

3. repairing the tail end;

4. adding a joint;

5. PCR amplification;

6. sequencing Illumina NovaSeq PE 150;

7. the bioinformation software HiCplotter makes a heat map.

As can be seen from the above experimental results (FIG. 3), the intensity of the interaction signal is diagonal, which indicates that the interaction inside the chromosome is stronger than the interaction between the chromosomes, and thus it can be seen that the rinsing method provided by the present invention does not damage the nuclear structure.

Example 2

This example provides an acanthus ilicifolius DNA extraction method, which is the same as that of example 1.

In this embodiment, the obtained acanthus ilicifolius DNA is further subjected to extraction amount detection and electrophoresis experiment. The total amount of extracted DNA detected by the method same as that of the example 1 is 2.78ug, the experimental result of the electrophoresis verification is shown in figure 4, and the specific process is as follows:

1. adding 50ul of 1.2x NEBuffer2 and 10ul of 20mg/ml proteinase K into the intermediate product before and after enzyme digestion in the experiment respectively, and digesting for 30min at 65 ℃;

4. taking a picture by using a gel imager;

from the above experimental results, it can be seen that the extraction amount of DNA is 2.78ug, which satisfies the Hi-C library building requirement; a complete genome strip exists before enzyme digestion (marker 2 in figure 4), and the extraction method of the invention does not damage the integrity of the genome; after enzyme digestion (marked 3 in figure 4), the bands are dispersed, which shows that the method removes impurities which affect the enzyme digestion reaction, so that the enzyme digestion reaction is efficient; after purification (labeled 1 in FIG. 4), a distinct ligation band was observed, indicating that the method of the present invention removes impurities affecting the ligation reaction, making the ligation reaction efficient.

Comparative example 1

This comparative example provides a method for extracting DNA from kelp, which is the same as that of example 1 except that the tissue sample to be tested is not rinsed with a rinsing solution.

As a result: the extracted DNA was highly viscous and pasty, and the total amount of extracted DNA was 7.6ug as determined by the QubitTM 4fluorometer (Invitrogen) in the same manner as in example 1, but was not suitable for Hi-C pooling due to the high viscosity.

Comparative example 2

This comparative example provides a method for extracting DNA from Laminaria japonica, which is the same as that of example 1 except that the rinsing solution was 100mM Tris-HCl, 0.35M sorbitol, 5mM EDTA, 1% PVP, and 1% β -mercaptoethanol.

This comparative example further carried out extraction amount detection and electrophoresis experiment on the obtained DNA. The total amount of extracted DNA detected by the method same as that of example 1 is 260ng, and the experimental results of electrophoresis verification are shown in FIG. 5, and the specific process is as follows:

4. taking a picture by using a gel imager;

as can be seen from the above experimental results, the extraction amount of DNA is 260ng, which does not meet the requirement of Hi-C library construction; there is a complete genome band before enzyme digestion (label 2 in FIG. 5), indicating that this extraction method does not destroy genome integrity; after enzyme digestion (labeled 3 in fig. 5), an enzyme digestion strip still exists, which indicates that the method cannot remove impurities such as polysaccharide and polyphenol which affect the enzyme digestion reaction, so that the enzyme digestion reaction is inefficient/ineffective; after purification (marked 1 in FIG. 5), there is a clear connecting band, which cannot indicate the high efficiency of the enzyme reaction, mainly because the genome is not cut due to the failure of the enzyme reaction.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims

1. A eukaryotic DNA extraction method suitable for Hi-C technology is characterized in that a sample tissue to be detected after cross-linking and wall breaking is rinsed by rinsing liquid and then cell nucleus extraction is carried out;

the rinsing liquid comprises glycerol, EDTA, PEG, PVP and β -mercaptoethanol.

2. The method as claimed in claim 1, wherein the rinsing solution comprises 100-120mM Tris-HCl, 5-10% (v/v) glycerol, 4-6mM EDTA, 5-10% (w/v) PEG, 0.8-1.2% (w/v) PVP, 0.8-1.2% (v/v) β -mercaptoethanol.

3. The method according to claim 1 or 2, wherein the molecular weight of the PEG is 4000-8000; the molecular weight of the PVP is 10000-40000.

4. The method according to any one of claims 1 to 3, wherein in the rinsing, the ratio of the sample tissue to be tested to the rinsing liquid is 1g (15-30) ml;

preferably, the number of rinsing is 2 to 5 times.

5. The method according to any one of claims 1 to 4, wherein the conditions of centrifugation in the rinsing are: 2500 + 3500g, 3.5-4.5 deg.C, 4-6 min.

6. The method of any one of claims 1 to 5, wherein after the extraction of cell nuclei, a second rinsing with the rinsing solution is performed.

7. The method of claim 6, wherein in the secondary rinsing, the ratio of the sample tissue to be detected to the rinsing liquid is 1g (4-6) ml;

preferably, the number of times of the secondary rinsing is 1 to 3 times.

8. The method according to claim 6 or 7, wherein in the secondary rinsing, the conditions of centrifugal separation are as follows: 1500-2000g, 3.5-4.5 deg.C, 4-6 min.

9. A eukaryotic DNA extraction rinsing liquid suitable for Hi-C technology is characterized in that the rinsing liquid comprises glycerol, EDTA, PEG, PVP and β -mercaptoethanol;

preferably, the rinsing liquid comprises 100-120mM Tris-HCl, 5-10% (v/v) glycerol, 4-6mM EDTA, 5-10% (w/v) PEG, 0.8-1.2% (w/v) PVP and 0.8-1.2% (v/v) β -mercaptoethanol;

more preferably, the molecular weight of the PEG is 4000-8000; the molecular weight of the PVP is 10000-40000.

10. Use of the method of any one of claims 1 to 8 or the rinse solution of claim 9 for three-dimensional conformation analysis of chromosomes, for mining gene regulatory sequences or for analysis of DNA interactions.