CN113846090A - Extraction method of high-quality enteromorpha DNA suitable for Pacbio three-generation long-reading long sequencing - Google Patents

Extraction method of high-quality enteromorpha DNA suitable for Pacbio three-generation long-reading long sequencing Download PDF

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CN113846090A
CN113846090A CN202111160712.6A CN202111160712A CN113846090A CN 113846090 A CN113846090 A CN 113846090A CN 202111160712 A CN202111160712 A CN 202111160712A CN 113846090 A CN113846090 A CN 113846090A
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毕远芝
何鹏
余小炜
李晖
赵仕兰
肖云平
王树伟
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Shanghai Oe Biotech Co ltd
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Abstract

The invention discloses an enteromorpha DNA extraction and purification method suitable for Pacbio long-reading sequencing. According to the invention, the enteromorpha genome DNA is extracted by CTAB and purified by combining magnetic beads, the purity of the extracted DNA is high, OD260/280 reaches 1.8-2.1, OD260/230 reaches 2.0-2.3, and the ratio of DNAqubit/Nanodrop obtained is 0.5-2. The length of the DNA fragment extracted by the invention is more than 50kb, the total amount reaches microgram level, and the Pacbio three-generation long-reading sequencing requirement can be met.

Description

Extraction method of high-quality enteromorpha DNA suitable for Pacbio three-generation long-reading long sequencing
Technical Field
The invention belongs to the field of DNA extraction, and relates to a method for extracting high-quality enteromorpha DNA suitable for Pacbio three-generation long-read long-sequence sequencing.
Background
The enteromorpha prolifera is rich in carbohydrates, proteins, crude fibers, amino acids, fatty acids, vitamins and various mineral substances, the carbohydrates are mainly polysaccharides, the enteromorpha prolifera polysaccharides are one of the main active ingredients of the enteromorpha prolifera, and the enteromorpha prolifera polysaccharides have multiple biological activities of immunoregulation, oxidation resistance, tumor resistance, blood fat reduction and the like. However, the large amount of the components exists in plants, the extraction solution is sticky during DNA extraction, the DNA and polysaccharide components are difficult to separate, the purity of the extracted DNA is poor, the content of the components such as polysaccharide is high, and great difficulty is brought to the subsequent Pacbio third-generation sequencing.
The PacBio-third generation sequencing refers to a single molecule sequencing technology, does not need to involve PCR amplification in the process of library construction and sequencing, and can be used for independently sequencing each DNA molecule. The method has the characteristics of super-long reading length, no need of template amplification, short running time and the like. The original DNA length directly determines the read length of the subreads obtained from the final sequencing. The method has the requirements of 1.8-2.1 for OD260/280 of DNA, 2.0-2.2 for 260/230, 0.5-2 for the ratio of the Qubit to the Nanodrop, and no tailing of a DNA main band, no RNA pollution, no protein pollution, no chelating agent pollution, no insoluble substances and the like, otherwise, the method has influence on a sequencing result, and even has no data in zero output.
A plant genome DNA extraction method, a sequencing method and an application patent (CN111909926A) were sent by Qingdahua Dageney research institute, the method adopts a CTAB method to extract plant genome DNA, then uses potassium acetate and absolute ethyl alcohol to incubate a supernatant to remove protein and polysaccharide, and combines with anion exchange resin to purify DNA solution, thereby obtaining the plant genome. Among them, the anion exchange resin used is QIAGEN Genomic-tip, which firstly has a high cost; secondly, a large number of test results show that the effect of the method for extracting the specified sample of the kit can reach the level of the specification, but the method for extracting the DNA of the enteromorpha sample can hardly reach the expected effect, protein pollution can occur in a glue hole during agarose electrophoresis as shown in CN111909926A figure 3, the purity of the DNA does not reach the standard, and the yield of the DNA is low.
In addition, many commercial DNA extraction kits are available in the market, and even if a Tiangen plant DNA extraction kit known in the industry is selected for testing, the extraction result does not meet the requirement of Pacbio third-generation sequencing.
The scheme of the Tiangen plant kit for extracting DNA from an enteromorpha sample comprises the following steps:
the method comprises the following operation steps:
1. taking about 100mg of fresh tissue or about 30mg of dry tissue of the plant, adding liquid nitrogen, and fully grinding.
2. The ground powder was quickly transferred to a centrifuge tube pre-filled with 700. mu.l of 65 ℃ pre-heated buffer GP1 (mercaptoethanol was added to pre-heated GP1 to a final concentration of 0.1% before the experiment), the mixture was quickly inverted and mixed, and then the centrifuge tube was placed in a 65 ℃ water bath for 20min, and the centrifuge tube was inverted during the water bath to mix the sample several times.
3. Add 700. mu.l chloroform, mix well and centrifuge at 12,000rpm (. about.13,400 Xg) for 5 min. Note: if plant tissue rich in polyphenol or starch is extracted, the same volume of phenol/chloroform/1: 1 is used for extraction before step 3.
4. Carefully transfer the upper aqueous phase obtained in the previous step into a new centrifuge tube, add 700. mu.l of buffer GP2, and mix well.
5. The mixed liquid was transferred to an adsorption column CB3, centrifuged at 12,000rpm (. about.13,400 Xg) for 30 seconds, and the waste liquid was discarded.
6. To adsorption column CB3, 500. mu.l of buffer GD was added, centrifuged at 12,000rpm for 30 seconds, the waste liquid was discarded, and adsorption column CB3 was put into the collection tube.
7. 600. mu.l of the rinsing solution PW was added to the adsorption column CB3, and centrifuged at 12,000rpm (. about.13,400 Xg) for 30 seconds, the waste liquid was discarded, and the adsorption column CB3 was put into the collection tube.
8. Operation 7 is repeated.
9. The adsorption column CB3 was returned to the collection tube, centrifuged at 12,000rpm for 2 minutes, and the waste liquid was discarded. The adsorption column CB3 was left at room temperature for several minutes to completely dry the residual rinse solution in the adsorption material.
10. The adsorption column CB3 is transferred into a clean centrifuge tube, 50-200 mu.l of elution buffer TE is suspended and dripped into the middle part of the adsorption membrane, the mixture is placed at room temperature for 2-5 minutes and centrifuged at 12,000rpm (13,400 Xg) for 2 minutes, and the solution is collected into the centrifuge tube.
The experimental results are as follows:
Figure BDA0003289856140000021
1. the DNA concentration is low;
2. the OD ratio of the DNA is poor;
3. the Q/N purity of the DNA was poor.
Disclosure of Invention
Aiming at the technical problems in the prior art, the invention provides a method for extracting enteromorpha DNA large fragment, which solves the following problems: the integrity of the DNA fragment extracted by the kit is not enough, and the DNA fragment can not reach the standard of third-generation sequencing long-reading and long-sequencing; DNA extracted by the kit has many impurities, insufficient purity and poorer OD ratio; the yield of enteromorpha DNA extracted by the kit is low and cannot reach the initial amount required by the third-generation sequencing.
In the prior art, in order to fully crack a sample in a short time, release more nucleic acid in cells and improve the yield of DNA, a sample-lysate mixture is uniformly mixed by using mechanical oscillation acting force in an experiment, but the operation easily breaks a DNA chain, the extracted nucleic acid fragment is short, and the integrity of the DNA is reduced; the requirement of long read length sequencing cannot be met. In order to increase the fragment size of the obtained DNA, the CTAB purification method provided by the invention is as follows: in the invention, the sample tube is flicked and uniformly mixed by fingers continuously in the incubation process to ensure that the sample is fully cracked, and the length of the extracted DNA fragment is more than 50kb, thereby ensuring the integrity of the DNA fragment. In addition, on one hand, the damage of the DNA in the organic reagent due to too long exposure time is avoided, on the other hand, the protein residue in a gel hole is avoided when the DNA is subjected to agarose gel electrophoresis detection due to too short standing time of the DNA in the organic reagent, and the purity of the DNA is reduced.
The invention provides a DNA extraction method suitable for Pacbio three-generation long-read-length sequencing, which comprises the following steps:
1: extraction of large-fragment enteromorpha DNA
1) Adding CTAB lysate and beta-mercaptoethanol into an EP tube, and then preheating at 55-70 ℃ (preferably 65 ℃);
in the step 1), the final concentration of the beta-mercaptoethanol in the CTAB lysate containing the beta-mercaptoethanol is 2-6%; preferably, it is 4%.
In the step 1), the CTAB lysate contains 2% -3% CTAB, 1-2M NaCl and 50-200mM Tris (pH8.0), 10-30mM EDTA; preferably, CTAB is 2%, NaCl is 1.5M, Tris (pH8.0) is 100mM, EDTA is 20 mM.
2) Pouring the ground enteromorpha powder into the preheated centrifugal tube in the step 1), and quickly shaking up the liquid in the centrifugal tube by hands;
3) adding protease K and RNase A into the solution obtained in the step 2), shaking the liquid in the tube by hand, and incubating the liquid in a constant-temperature metal bath at 55-70 ℃ (preferably 65 ℃) for 30-90 minutes (preferably 60 minutes);
in the step 3), the volume of the proteinase K is 10-30 μ l, and the volume of the RNase is: 3-6 mul; preferably 20. mu.l and 4. mu.l, respectively.
In the step 3), the proteinase K is a strong serine protein lytic enzyme which is separated from candida albicans and has wide cleavage activity, and is used for cleaving carboxyl terminal peptide bonds of aliphatic amino acids and aromatic amino acids. The activity of RNase and DNase was removed by purification. The enzyme has activity in a wide pH range (4-12.5) and at a high temperature (50-70 ℃), can stably exist in urea and SDS and still has the capability of degrading natural protein. Used for separating plasmid or genome DNA and RNA. In DNA extraction, the main role is to enzymatically cleave histone bound to nucleic acid, to free DNA in solution, followed by extraction by various methods, to remove impurities, and to collect DNA. EDTA and other chelating agents cannot deactivate them. The stock solution is generally 10mg/ml or 20 mg/ml. Normally, the protease K solution is colorless and transparent.
In the step 3), the RNase is RNase A which is endoribonuclease from bovine pancreas and can specifically attack the 3 ' end of the pyrimidine residue on RNA to cut the phosphodiester bond formed by cytosine or uracil and adjacent nucleotide, and the final products of the reaction are 3 ' pyrimidine nucleotide and oligonucleotide with 3 ' pyrimidine nucleotide at the tail end. In the absence of cofactors and divalent cations, the action of ribonuclease A can be inhibited by placental RNase inhibitor (RNase) or vanadyl-ribonucleoside complex. The reaction conditions of RNase A are extremely wide and difficult to inactivate. Under low salt concentration (0-100 mmol/l NaCl), RNA in a single-stranded RNA, double-stranded RNA and DNA: RNA hybrid is cut by RNase A; RNase A specifically cleaves single-stranded RNA at NaCl concentrations of 300mM or higher.
4) Cooling, then adding a precooled volume ratio of 25: 24: 1 phenol: chloroform: the isoamyl alcohol is fully shaken by hands and then is placed at 0-8 ℃ (preferably 4 ℃) for 1-15 minutes (preferably 10 minutes);
in step 4), the phenol is tris saturated phenol with pH 8.0.
5) Centrifuging to obtain a supernatant, and adding a solvent with a volume ratio of 24: 1 chloroform: isoamyl alcohol, shaking up by hand, placing at 0-8 deg.C (preferably 4 deg.C) for 1-30 min (preferably 10 min);
the centrifugation conditions were: 12000-14000rpm (preferably 13000rpm), 0-6 deg.C (preferably 4 deg.C), and low-temperature centrifugation for 5-30 minutes (preferably 15 minutes).
6) Centrifuging to obtain supernatant, adding 0.5-1 (preferably 0.5) times of isopropanol, mixing by turning the centrifugal tube slightly and reversely, and freezing at-20 deg.C to-80 deg.C (preferably-80 deg.C) for 10 min to 2 hr (preferably 30 min);
the centrifugation conditions were: 12000-14000rpm (preferably 13000rpm), 0-6 deg.C (preferably 4 deg.C), and low-temperature centrifugation for 5-30 minutes (preferably 15 minutes).
7) Centrifuging, washing the precipitate with 70% ethanol, centrifuging, and removing supernatant to obtain large fragment DNA; after the DNA is dried, the DNA is dissolved with an elution buffer.
The centrifugation conditions were: 12000-14000rpm (preferably 13000rpm), 0-6 deg.C (preferably 4 deg.C), and low-temperature centrifugation for 5-30 minutes (preferably 15 minutes).
The large fragment DNA refers to DNA with the fragment length of more than 50 kb; preferably, it is 50kb to 70kb of DNA.
2: DNA purification by diluting DNA to an appropriate concentration with magnetic beads
8) Adding 0.6 multiplied purified magnetic beads into enteromorpha DNA with the concentration lower than 50ng/ul, lightly and uniformly flicking, and standing for 10 minutes at room temperature.
9) Adsorbing on magnetic frame for 5min, removing supernatant, washing with 70% ethanol for 10 s, removing supernatant, washing for 2 times, removing residual ethanol, and taking off the centrifuge tube from magnetic frame.
10) And eluting the DNA by using an elution buffer solution, standing at room temperature for 2 minutes, placing on a magnetic frame for adsorption for 5 minutes, and then sucking the supernatant and placing in a new centrifuge tube to obtain the enteromorpha DNA.
The purity of the enteromorpha DNA is as follows: no protein residue exists in the gel hole, OD260/280 reaches 1.8-2.1, OD260/230 reaches 2.0-2.3, and the ratio of DNAqubit/Nanodrop obtained is 0.5-2.
The extraction yield of the enteromorpha DNA is improved from the high-quality DNA about 2 mu g obtained from the original 50mg sample to the high-quality DNA about 10 mu g, the yield is improved by about 5 times, and the Pacbio three-generation long-reading sequencing requirement can be met.
Further, the method further comprises step 11): repeating the steps 8) -9) for 1 time to ensure that the DNA achieves high purity: OD260/280 reaches 1.8-2.1, OD260/230 reaches 2.0-2.3, and the ratio of the obtained DNA Qubit/Nanodrop is 0.5-2.
In the present invention, the elution buffer is diluted to a DNA concentration of less than 50 ng/. mu.l, so that the subsequent experiment can be started.
In a specific embodiment, the DNA extraction method suitable for Pacbio three-generation long read length sequencing specifically comprises the following steps:
1: extraction of large-fragment enteromorpha DNA
1) A centrifuge tube was taken, 600. mu.l of 2-4% (preferably 2%) CTAB lysate and 20. mu.l of beta-mercaptoethanol were added, and after mixing, the tube was preheated in a 65 ℃ metal bath.
2) And pouring liquid nitrogen into the grinding cylinder, and precooling the cylinder body.
3) Pouring the enteromorpha sample into liquid nitrogen, and quickly grinding the enteromorpha sample by using a grinding rod until the enteromorpha sample is powdered.
4) About 200mg of enteromorpha powder is poured into 620 mu l of 2% CTAB mixed lysate, and the liquid in the tube is quickly shaken up by hands.
5) Add 20. mu.l proteinase K, 4. mu.l RNase A, shake the tube liquid by hand and incubate it for 1 hour in a constant temperature metal bath at 65 ℃.
6) The centrifuge tube was removed from the metal bath, cooled at 4 ℃ for 5 minutes and 500. mu.l of pre-cooled phenol: chloroform: isoamyl alcohol (volume ratio 25: 24: 1), shaking thoroughly by hand, standing at 4 deg.C for 10 minutes.
7) The tube is centrifuged at 12000-14000rpm (preferably 13000rpm) and 0-6 deg.C (preferably 4 deg.C) for 5-30 minutes (preferably 15 minutes) to obtain a supernatant.
8) Chloroform was added to the supernatant: isoamyl alcohol (volume ratio 24: 1), hand shake, and standing at 0-6 deg.C (preferably 4 deg.C) for 10 minutes.
9) The tube was centrifuged at 13000rpm at 4 ℃ for 10 minutes to obtain a supernatant.
10) To the supernatant was again added 500. mu.l chloroform: isoamyl alcohol (24: 1), shaking by hand, standing at 4 ℃ for 10 minutes.
11) Centrifuging at 13000rpm at 4 deg.C for 10 min, collecting supernatant, adding 0.5 times of isopropanol, mixing by turning the tube by hand, and freezing at-80 deg.C for half an hour.
12) The tube was centrifuged at 13000rpm at 4 ℃ for 15 minutes, the supernatant was removed, and the precipitate was retained.
13) The precipitate was washed with 70% ethanol, centrifuged at 13000rpm at 4 ℃ for 5 minutes, and the supernatant was removed.
14) Repeating the step 13)1 time.
15) After it has dried, the DNA is dissolved in an elution buffer.
2: DNA purification by diluting DNA to an appropriate concentration with magnetic beads
1) Subsequent experiments can be started by dilution with elution buffer to a DNA concentration below 50 ng/. mu.l.
2) The purified beads were taken out 30 minutes in advance and equilibrated at room temperature.
3) Adding purified magnetic beads (0.4-0.7 times, preferably 0.6 times) into Enteromorpha DNA with concentration lower than 50ng/μ l, gently and uniformly flicking, and standing at room temperature for 10 min.
4) Adsorbing on magnetic frame for 5min, removing supernatant, washing with 70% ethanol for 10 s, removing supernatant, washing for 2 times, removing residual ethanol, and taking off the centrifuge tube from magnetic frame.
5) Eluting DNA with elution buffer, standing at room temperature for 2 min, adsorbing on magnetic frame for 5min, and sucking supernatant and placing in new centrifuge tube.
6) Repeating the steps 2) -4)1 time to obtain high-purity DNA.
The invention also provides a large-fragment enteromorpha DNA extracted by the method.
The large-fragment enteromorpha DNA refers to enteromorpha DNA with the fragment length of more than 50 kb; preferably, the DNA is 50kb to 70 kb.
The large-fragment enteromorpha DNA is suitable for Pacbio three-generation long-reading long sequencing.
The invention also provides a DNA extraction kit, which comprises lysate, protease K and RNase A.
The lysis solution comprises 2-3% CTAB, 1-2M NaCl, 50-200mM Tris (pH8.0), 10-30mM EDTA, and 4-6% beta-mercaptoethanol is added before use.
The contents of the protease K and the RNase A in the DNA extraction kit are respectively 20 mul/sample and 4 mul/sample.
The invention also provides application of the DNA extraction kit in DNA extraction.
The innovation points of the invention are as follows:
(1) a violent shaking method is not adopted in the extraction process, so that the mechanical shearing force on the DNA is reduced, and the length of the obtained DNA fragment is increased.
(2) The extraction is more complete by prolonging the action time in the multi-step organic reagent treatment process.
(3) Protease K and RNase A are added into the extracted lysate to remove protein and RNA pollution and improve the purity of DNA.
(4) And the obtained DNA is purified by adopting 0.6 multiplied by magnetic beads, so that low-quality small-fragment DNA, protein and a small amount of residual polysaccharide are removed, and the purity of the finally obtained DNA is improved.
The beneficial effects of the invention include: compared with the prior art in which a kit method is used for extraction, the method provided by the invention has the advantages that the extraction is obviously improved in many aspects: improvement of DNA integrity: the main belt is single and has no tail; purity of DNA: no protein residue exists in the rubber pores, OD260/280 reaches 1.8-2.1, OD260/230 reaches 2.0-2.3, the ratio of the obtained DNA Qubit/Nanodrop is 0.5-2, most of the DNA Qubit/Nanodrop is about 1.0, and the ratio of the DNA Qubit/Nanodrop in the prior art is only 0.3; the DNA extraction yield is improved from the high-quality DNA about 2 mu g obtained by the original 50mg sample to the high-quality DNA about 10 mu g, the yield is improved by about 5 times, and the requirement of Pacbio three-generation long-reading sequencing can be met.
Drawings
FIG. 1 is an electrophoretogram of DNA extracted by the method of example 1 of the present invention.
FIG. 2 is an electrophoretogram of DNA extracted by the method of example 2 of the present invention.
FIG. 3 is an electrophoretogram of DNA extracted by the method of example 3 of the present invention.
Detailed Description
The present invention will be described in further detail with reference to the following specific examples and the accompanying drawings. The procedures, conditions, experimental methods and the like for carrying out the present invention are general knowledge and common general knowledge in the art except for the contents specifically mentioned below, and the present invention is not particularly limited.
Example 1
Procedure for the preparation of the
Firstly, preparation before experiment:
consumable material: grinding cylinder, grinding rod, liquid nitrogen, centrifuge tube, pipette gun, etc.;
equipment: metal bath, low-temperature centrifuge, ultra-low temperature refrigerator at-80 ℃, pulse field electrophoresis system, Nanodrop2000, qubit3.0 and the like;
reagent: 2% CTAB lysate, 70% ethanol, beta-mercaptoethanol, protease K, RNA enzyme A, purified magnetic beads, etc.
II, an experiment step:
1: extraction of large fragment DNA
1) The centrifuge tube was taken, 600. mu.l of 2% CTAB lysate and 20. mu.l of beta-mercaptoethanol were added, mixed and placed in a 65 ℃ metal bath for preheating.
2) And pouring liquid nitrogen into the grinding cylinder, and precooling the cylinder body.
3) Pouring the enteromorpha sample into liquid nitrogen, and quickly grinding the enteromorpha sample by using a grinding rod until the enteromorpha sample is powdered.
4) About 200mg of enteromorpha powder is poured into 620 mu l of 2% CTAB mixed lysate, and the liquid in the tube is quickly shaken up by hands.
5) Add 20. mu.l proteinase K, 4. mu.l RNase A, shake the tube liquid by hand and incubate it for 1 hour in a constant temperature metal bath at 65 ℃.
6) Taking out the centrifuge tube from metal bath, cooling at 4 deg.C for 5min, adding 500 μ l precooled phenol: chloroform: isoamyl alcohol (25: 24: 1), shaking thoroughly with hand, and standing at 4 deg.C for 10 min.
7) The tube was centrifuged at 13000rpm at 4 ℃ for 15 minutes to obtain a supernatant.
8) Chloroform was added to the supernatant: isoamyl alcohol (24: 1), shaking by hand, standing at 4 deg.C for 10 min.
9) The tube was centrifuged at 13000rpm at 4 ℃ for 10 minutes to obtain a supernatant.
10) Adding 500 μ l chloroform/isoamyl alcohol (24: 1) into the supernatant, shaking by hand, and standing at 4 deg.C for 10 min.
11) Centrifuging at 13000rpm at 4 deg.C for 10 min, collecting supernatant, adding 0.5 times of isopropanol, mixing by turning the tube by hand, and freezing at-80 deg.C for half an hour.
12) The tube was centrifuged at 13000rpm at 4 ℃ for 15 minutes, the supernatant was removed, and the precipitate was retained.
13) The precipitate was washed with 70% ethanol, centrifuged at 13000rpm at 4 ℃ for 5 minutes, and the supernatant was removed.
14) Repeating the step (13)1 time.
15) After it is dried, the DNA is dissolved with an eluent.
2: DNA purification by diluting DNA to an appropriate concentration with magnetic beads
1) Subsequent experiments can be started by diluting the eluate to a DNA concentration below 50 ng/. mu.l.
2) The purified beads were taken out 30 minutes in advance and equilibrated at room temperature.
3) Adding 0.6 multiplied purified magnetic beads into enteromorpha DNA with the concentration lower than 50 ng/mu l, lightly and uniformly flicking, and standing for 10 minutes at room temperature.
4) Adsorbing on magnetic frame for 5min, removing supernatant, washing with 70% ethanol for 10 s, removing supernatant, washing for 2 times, removing residual ethanol, and taking off the centrifuge tube from magnetic frame.
5) Eluting DNA with the eluent, standing at room temperature for 2 minutes, placing on a magnetic frame for adsorption for 5 minutes, and then sucking the supernatant and placing in a new centrifuge tube.
6) And (5) repeating the step (2-4) for 1 time to ensure that the DNA achieves high purity.
Thirdly, experimental results:
1) the DNA was subjected to quality inspection using both Nanodrop and Qubit quality inspection tools, and the results are shown in the following table
Figure BDA0003289856140000091
The concentration results show that:
a) the 260/680 and 260/230 ratios of the DNA both meet the technical requirements of Pacbio sequencing;
b) the ratio of the Qubit/Nano of the DNA is close to 1.0, and the closer to 1.0, the higher the quality of the subsequent library is;
c) the total amount of DNA reaches more than 14 mu g, and the construction of a Pacbio sequencing large fragment library is satisfied.
2) The DNA was subjected to agarose gel electrophoresis, and the results are shown in FIG. 1.
The banding results show that:
a) the DNA band of the CTAB purification method is single and has no tailing, and long fragment DNA meeting the technical requirements of Pacbio sequencing is obtained;
b) DNA extracted by the kit method has a main band and an obvious tailing, is dragged to be below 2kb, and cannot meet the large-fragment library construction technology of the Pacbio sequencing technology.
Example 2
The specific experimental conditions are the same as those in the embodiment 1 of the invention, and under the condition that other experimental conditions are not changed, the protein pollution in the glue hole is found through electrophoresis detection without using proteinase K. (FIG. 2)
Example 3
The specific experimental conditions are the same as those in example 1 of the invention, and RNA residue is found by electrophoresis detection without using RNase A under the condition that other experimental conditions are not changed. (FIG. 3)
The protection of the present invention is not limited to the above embodiments. Variations and advantages that may occur to those skilled in the art may be incorporated into the invention without departing from the spirit and scope of the inventive concept, which is set forth in the following claims.

Claims (10)

1. A DNA extraction method suitable for Pacbio three-generation long-read-length sequencing is characterized by comprising the following steps:
(1) extraction of large-fragment enteromorpha DNA
1) Adding CTAB lysate and beta-mercaptoethanol into a centrifugal tube, and then preheating at 55-70 ℃;
2) pouring the enteromorpha powder ground by liquid nitrogen into the preheated centrifugal tube in the step 1), and quickly shaking up the liquid in the centrifugal tube by hands;
3) adding protease K and RNase A into the step 2), shaking the liquid in the tube uniformly by hands, and placing the tube in a constant-temperature metal bath at the temperature of 55-70 ℃ for incubation for 30-90 minutes;
4) cooling, then adding a precooled volume ratio of 25: 24: 1 phenol: chloroform: shaking isoamyl alcohol, standing at 0-8 deg.C for 1-15 min;
5) centrifuging to obtain a supernatant, and adding a solvent with a volume ratio of 24: 1 chloroform: shaking isoamyl alcohol, standing at 0-8 deg.C for 1-30 min;
6) centrifuging to obtain a supernatant, adding isopropanol with the volume of 0.5-1 time of that of the supernatant, slightly and evenly reversing the centrifugal tube by hands, and freezing at the temperature of-20 to-80 ℃ for 10 minutes to 2 hours;
7) centrifuging, washing the precipitate with 70% ethanol, centrifuging, and removing supernatant to obtain large-fragment Enteromorpha DNA; after the enteromorpha DNA is dried, dissolving the enteromorpha DNA by using an elution buffer solution;
(2) DNA dilution and DNA purification with magnetic beads
8) Adding 0.6 multiplied purified magnetic beads into the enteromorpha DNA with the concentration lower than 50 ng/mu l in the step 7), and standing for 10 minutes at room temperature;
9) then placing the centrifuge tube on a magnetic frame for adsorption for 5 minutes, removing the supernatant, washing the centrifuge tube for 10 seconds by using 70% ethanol, removing the supernatant, washing the centrifuge tube for 2 times, removing residual alcohol, and taking down the centrifuge tube from the magnetic frame;
10) and eluting the enteromorpha DNA by using an elution buffer solution, standing at room temperature for 2 minutes, placing on a magnetic frame for adsorption for 5 minutes, and then sucking the supernatant and placing in a new centrifugal tube to obtain the enteromorpha DNA.
2. The method of claim 1, wherein in step 1), the final concentration of β -mercaptoethanol in the β -mercaptoethanol-containing CTAB lysate is from 2% to 6%.
3. The method of claim 1, wherein in step 1), the CTAB lysate comprises 2% -3% CTAB, 1-2M NaCl and 50-200mM Tris (ph8.0), 10-30mM EDTA.
4. The method according to claim 1, wherein in step 3), the volume of proteinase K is 10-30 μ l; the volume of the RNase is 2-6 μ l;
and/or, in the step 4), the phenol is tris saturated phenol with pH 8.0.
5. The method of claim 1, wherein the centrifugation conditions of steps 5) -7) are all as follows: 12000-14000rpm, and low-temperature centrifugation at 0-6 ℃ for 5-30 minutes.
6. The method of claim 1, wherein in the step 7), the large-fragment enteromorpha DNA refers to enteromorpha DNA with a fragment length of more than 50 kb.
7. A DNA extraction kit is characterized by comprising lysate, protease K and RNase A.
8. The kit of claim 7, wherein the lysis solution comprises 2% to 3% CTAB, 1-2M NaCl and 50-200mM Tris (ph8.0), 10-30mM EDTA, with 2% to 6% β -mercaptoethanol added prior to use.
9. The kit of claim 7, wherein the contents of proteinase K and RNaseA in the DNA extraction kit are 10-30. mu.l/sample and 2-6. mu.l/sample, respectively.
10. Use of the DNA extraction kit of any one of claims 7-9 for extracting DNA.
CN202111160712.6A 2021-09-30 2021-09-30 Extraction method of high-quality enteromorpha DNA suitable for Pacbio three-generation long-reading long sequencing Pending CN113846090A (en)

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Application publication date: 20211228