CN111607636A

CN111607636A - Method for extracting high-quality DNA from red tide heterocurvulus cells

Info

Publication number: CN111607636A
Application number: CN202010597957.4A
Authority: CN
Inventors: 胡广伟; 张珍珍; 姬南京; 申欣; 蔡月凤; 覃昊; 石一彪; 邓圣贤
Original assignee: Jiangsu Ocean University
Current assignee: Jiangsu Ocean University
Priority date: 2020-06-28
Filing date: 2020-06-28
Publication date: 2020-09-01

Abstract

The invention discloses a method for extracting high-quality DNA from heterocurvulus akashiwo cells, which comprises the following steps: culturing algae cells; screening algae cell lysis buffer solution; the collection method of the algae cells is groped; the method mainly comprises the steps of collecting heterocurvula akashiwo cells by using a filter membrane and selecting a proper CTAB cell lysate, and the result shows that the genomic DNA of the heterocurvula akashiwo extracted by using the novel method is superior to other methods in quality and integrity, and is suitable for PCR analysis and DNA high-throughput sequencing.

Description

Method for extracting high-quality DNA from red tide heterocurvulus cells

Technical Field

The invention relates to the field of DNA extraction of Heterocurus akashiwo, in particular to a method for extracting high-quality DNA from Heterocurus akashiwo cells.

Background

Harmful algal blooms are formed as a result of explosive proliferation of certain algae, and can cause significant damage to marine ecosystems and local economies. In recent years, the frequency, intensity and geographical distribution of harmful algal blooms have increased around the world. The red tide heterosigma akashiwo is a typical red tide species, is a wide-temperature and wide-salinity organism and is distributed in a plurality of sea areas in the world. This alga has been studied extensively because it produces fish toxins when red tides develop, severely damaging the marine ecosystem, and causing death of a large number of artificially cultivated fish species. However, the mechanisms of algal bloom formation and toxin production are still unclear, and information on the molecular mechanism or biochemical processes regulating the growth and metabolism of the species is very important in order to better understand how algal blooms are formed from red tide heterocurvula and how fish are killed when algal blooms are formed.

DNA-based molecular biology methods are one of the effective methods for studying biological characteristics. For molecular analysis, genetic engineering, such as cloning and DNA sequencing, the initial step of all these steps is DNA extraction. There are many DNA extraction methods such as Sodium Dodecyl Sulfate (SDS) method, cetyltrimethylammonium bromide (CTAB) method and commercialized DNA extraction kits. However, some of the DNA extraction methods described above may be applicable to certain specific organisms, but not to specific algae species. The genome of the Heterocurus akashiwo is large, and complete genome DNA is difficult to obtain, so that obtaining the genome of the Heterocurus akashiwo with high concentration, high purity and high integrity is a prerequisite for developing a high-throughput sequencing technology of the DNA of the species.

The difficulty in extracting DNA from plants and algae is that they have a unique cellular structure. The cells of Heterocurus akashiwo are fragile and have no cell wall. Therefore, when the algal cells are collected by centrifugation, the cells are aggregated to form a cell mass, which makes it more difficult to completely lyse the algal cells. Although homogenizers can damage the structure of cells, physical manipulation can reduce the integrity of genomic DNA, making DNA obtained by this method unsuitable for downstream experiments such as genome sequencing.

Disclosure of Invention

The invention aims to provide a method for extracting high-quality DNA from heterocurvulus akashiwo cells aiming at the defects of the prior art so as to solve the problems in the background technology.

In order to achieve the purpose, the invention provides the following technical scheme: a method for extracting high-quality DNA from heterocurvulus akashiwo cells comprises the following steps:

s1 culture of algae cells: filtering seawater for preparing algae culture solution with 0.22 μm filter membrane, and autoclaving to obtain algae culture solution with salinity of 30 PSU;

s2 screening of algal cell lysis buffer: obtaining high-quality DNA by an SDS treatment method and a CTAB extraction method; firstly, obtaining algae cells from a culture medium, and then suspending cell particles in SDS and CTAB cell lysate respectively;

s3, collecting algae cells: using a 0.3 μm PVDF filter membrane, then transferring the filter membrane to 0.8mL CTAB cell lysate (2% CTAB,100mm tris-hci ph8.0,1.4m nacl, 0.2% beta mercaptoethanol, 0.1mg/mL proteinase K) preheated at 60 ℃, cutting the filter membrane with scissors, sealing the tube orifice of the centrifuge tube with a sealing membrane, incubating at 60 ℃ for 2 hours, mixing gently by inverting from time to time, centrifuging the sample at 12000rpm for 15s, and transferring the cell lysate to a new centrifuge tube;

s4, adding 800 mu L of chloroform into the cell sap, slightly inverting the centrifuge tube for 2 minutes to mix the mixture evenly, standing the mixture for 2 minutes at room temperature, and then centrifuging the mixture for 10 minutes at 12000rpm at 4 ℃;

s5, transferring the upper aqueous phase into a new centrifuge tube, adding RNase, and incubating at 37 ℃ for 30 minutes;

s6, adding equal volume of chloroform into the mixed solution after warm bath, and centrifuging at 12000rpm for 10 minutes;

s7, transferring the upper aqueous phase into another new centrifuge tube, adding 1/10 volumes of sodium acetate and 2/3 volumes of isopropanol, and slightly reversing and mixing;

s8, standing at room temperature for 10 minutes, standing at 4 ℃, 12000rpm, and centrifuging for 5 minutes. Removing supernatant, washing with 70% anhydrous ethanol for 2 times, sucking off anhydrous ethanol, and air drying at room temperature; finally, the dried DNA sample was resuspended in 100. mu.L of sterile water and subjected to electrophoresis using 0.6% agarose gel;

s9 analysis of DNA concentration, purity and integrity: the DNA concentration was quantitatively determined by means of an ultramicro spectrophotometer.

In a preferred embodiment of the present invention, the culture medium for culturing algal cells in S1 is f/2 medium without adding si, and the algal cells are cultured under a culture condition at 20 ℃, and the cell concentration is calculated after counting the cells under a microscope using a Sedgwick-Rafter counting plate.

As a preferred technical scheme of the invention, the calculation formula of the algae cell concentration is as follows: c_ADR G50, wherein C_AIndicating the concentration of the algae cells; DR represents the dilution factor of the algae culture solution during the algae cultivation; g represents the average value of the number of algal cells.

In a preferred embodiment of the present invention, the step S2 of lysing algal cells with SDS comprises:

s2-1-1: collecting algae cells by centrifugation, suspending cell particles in SDS cell lysate (0.1MEDTA, 1% SDS), and incubating the cell suspension at 56 ℃ overnight;

s2-1-2: adding chloroform with the same volume into the cell suspension, repeatedly reversing and uniformly mixing, centrifuging at 12000rpm at 4 ℃ for 10 minutes, carefully transferring the upper aqueous phase into a new centrifuge tube, and adding 1/10 volumes of NaAc and 2/3 volumes of isopropanol;

s2-1-3: the mixed cell suspension is kept still for 10 minutes at room temperature, then centrifuged for 5 minutes at 4 ℃ and 12000rpm, DNA is collected, washed twice by 70% absolute ethyl alcohol and dried, and then the DNA is dissolved in sterile water.

In a preferred embodiment of the present invention, the step S2 of disrupting algal cells using CTAB comprises the following steps:

s2-2-1: incubating at 55 ℃ for 5 hours in 0.5ml of DNA lysis buffer (10mM Tris, pH 8.0; 100mM EDTA, pH 8.0; 0.5% SDS and 200. mu.g/ml proteinase K); 82.5. mu.L of 5M NaCl was added to a final concentration of 0.7M;

s2-2-2: then 82.5. mu.L of pre-warmed 10% (w/v) CTAB solution was added to the cell lysate, vortexed for 1 minute and incubated at 56 ℃ for 10 minutes;

s2-2-3: add 600. mu.L of chloroform at 12000rpm, centrifuge for 10 minutes, transfer the supernatant to a new centrifuge tube, then add 2 volumes of DNA binding buffer to each DNA sample, and the rest of the procedure is the same as for the DNA purification and concentration kit of Zymo.

In a preferred embodiment of the present invention, the method for determining the DNA concentration in S9 comprises: the purity of the nucleic acid was quantitatively determined by Nanodrop based on absorbance values at 230, 260 and 280nm and the ratio of 260/280 to 260/230 was calculated, the genomic DNA was extracted by the optimized method, the integrity thereof was identified by 0.6% agarose gel electrophoresis, and the DNA bands were observed by gel nucleic acid staining.

The invention has the beneficial effects that: the invention provides a method for extracting high-quality DNA from heterocurvulus akashiwo cells, which mainly adopts a method for collecting the heterocurvulus akashiwo cells by using a filter membrane and selects proper CTAB cell lysate. The results show that: the new method is superior to other methods in the concentration and quality of the extracted genome DNA of the Heterocurus akashiwo, and is suitable for PCR analysis and DNA high-throughput sequencing.

Drawings

FIG. 1 is a flow chart of the present invention;

FIG. 2 standard electrophoresis analysis of the integrity and yield of genomic DNA extracted using different methods;

FIG. 3 standard electrophoresis method for DNA integrity;

FIG. 4 evaluation of DNA quality by standard electrophoresis.

Detailed Description

The following detailed description of the preferred embodiments of the present invention is provided to enable those skilled in the art to more readily understand the advantages and features of the present invention and to clearly define the scope of the invention.

Example 1: referring to fig. 1, the present invention provides a technical solution: a method for extracting high-quality DNA from heterocurvulus akashiwo cells comprises the following steps: the invention has the following implementation steps:

the method comprises the following steps:

the culture medium for culturing the algae cells is f/2 culture medium without adding silicon element, culturing is carried out under the culture condition of 20 ℃, the concentration of the algae cells is counted under a microscope by using a Sedgwick-Rafter counting plate, and then the cell concentration is calculated.

The calculation formula of the algae cell concentration is as follows: c_ADR G50, wherein C_AIndicating the concentration of the algae cells; DR represents the dilution factor of the algae culture solution during the algae cultivation; g represents the average value of the number of algal cells.

the specific steps for lysing algal cells using SDS:

s2-1-1: collecting algal cells by centrifugation, suspending the cell particles in SDS buffer (0.1MEDTA, 1% SDS), and incubating the cell suspension at 56 ℃ overnight;

s2-1-2: an equal volume of chloroform was added to the cell lysate, mixed by gentle inversion, and centrifuged at 12000rpm for 10 minutes at 4 ℃. Carefully transfer the upper aqueous phase to a new centrifuge tube and add 1/10 volumes of NaAc and 2/3 volumes of isopropanol;

s2-1-3: the mixed cell lysate was allowed to stand at room temperature for 10 minutes, and then centrifuged at 12000rpm at 4 ℃ for 5 minutes to collect DNA. The DNA was washed twice with 70% absolute ethanol, air-dried, and then dissolved in sterile water.

Specific steps for disrupting algal cells using CTAB:

s2-2-2: then 82.5. mu.L of pre-preheated 10% (w/v) CTAB solution was added to the lysate, vortexed for 1 minute and then incubated at 56 ℃ for 10 minutes;

s2-2-3: add 600. mu.L of chloroform at 12000rpm, centrifuge for 10 minutes, transfer the upper aqueous phase to a new centrifuge tube, then add 2 volumes of DNA binding buffer to each DNA sample, and the rest of the procedure is the same as for Zymo's DNA purification and concentration kit.

S3, collecting algae cells: using 0.3 μm PVDF filter membrane, then transferring the filter membrane into 0.8mL CTAB buffer (2% CTAB,100mM Tris-HClpH8.0,1.4MNaCL, 0.2% beta mercaptoethanol, 0.1mg/mL proteinase K) preheated at 60 ℃, cutting the filter membrane with scissors, sealing the bottle cap with a sealing membrane, incubating at 60 ℃ for 2 hours, gently inverting and mixing from time to time, centrifuging the sample at 12000rpm for 15s, and transferring the cell lysate into a new centrifuge tube;

s4, adding 800 mu L of chloroform into the cell lysate, slightly inverting the centrifuge tube for 2 minutes to mix the mixture evenly, standing the mixture for 2 minutes at room temperature, and then centrifuging the mixture for 10 minutes at 12000rpm at 4 ℃;

s5, transferring the upper aqueous phase to a new centrifuge tube, adding RNase, and carrying out warm bath at 37 ℃ for 30 minutes;

s6, adding equal volume of chloroform into the cell lysate after the temperature bath, and centrifuging at 12000rpm for 10 minutes;

s8, standing at room temperature for 10 minutes at 4 ℃, 12000rpm, centrifuging for 5 minutes, and removing the supernatant. Washing the precipitate with 70% anhydrous ethanol for 2 times, sucking off the anhydrous ethanol, and air drying at room temperature; finally, the dried DNA sample was resuspended in 100. mu.L of sterile water and subjected to electrophoresis using 0.6% agarose gel;

s9 analysis of DNA concentration, purity and integrity: the DNA concentration was quantitatively determined using a ultramicrospectrophotometer. The purity of nucleic acid was quantitatively determined by Nanodrop based on absorbance values at 230, 260 and 280nm and calculated to 260/280 and 260/230 values, and genomic DNA of Heterocurus akashiwo was extracted by the optimized method, the integrity thereof was identified by 0.6% agarose gel electrophoresis, and DNA bands were observed by gel nucleic acid staining.

Results 1: integrity of DNA

As shown in FIG. 2, DNA was extracted by SDS method and SDS method, and then DNA was purified by elution column and DNA was extracted by conventional laboratory method to obtain genomic DNA of Heterocurus akashiwo, and the results of gel electrophoresis were analyzed. DNA extracted from Heterocurus akashii by SDS showed a strong band (FIG. 2, band 1). When DNA was extracted by SDS method and purified by Zymo purification kit, the band was found to be weak (FIG. 2, band 2). The agarose gel electrophoresis results showed that the DNA obtained using standard DNA extraction methods was band-dispersed after electrophoresis (FIG. 2, band 3). By analyzing the 260/280 and 260/230 values of the DNA of the red tide bent algae extracted by the three methods and the integrity of the obtained DNA, the obtained DNA is found to have low purity or low integrity. In addition, the harvesting method of algae cells also affects the concentration of DNA, the algae cells collected by a filter membrane method show a strong band (FIG. 4, band 1), which indicates that the concentration of the extracted DNA is high, while the cells collected by centrifugation are crushed by using magnetic beads, and the electrophoresis result of the extracted DNA shows that the DNA band is dispersed (FIG. 4, band 2), which indicates that the integrity of the extracted DNA is low. Comparing the quality of DNA extracted by the CTAB method and the SDS method shows that the DNA extraction by the CTAB method can reduce the pollution of protein, which indicates that the CTAB method is more suitable for extracting the genome DNA of the Heterosigma akashiwo.

Results 2: purity of DNA

Nucleic acid purity was assessed by calculating 260/280 and 260/230 values, 260/280 as an indicator of the presence or absence of protein contamination, and 260/230 as a ratio to monitor other contaminants such as carbohydrates, guanidine thiocyanate and phenols. The 260/280 value for DNA extracted by SDS method was 1.66, the 260/230 value was 0.94 (Table 1, Lane 1); when the isolated DNA was further purified using a DNA purification column, the ratios of 260/280 and 260/230 were increased to 1.75 and 1.59, respectively (Table 1, Lane 2). In contrast, the DNA extracted using the standard method had 260/280 and 260/230 values of 1.83 and 2.03, respectively (table 1, Lane 3). The DNA extracted by CTAB method showed better 260/280 and 260/230 values (Table 2, Line1, 2) than 260/280 and 260/230 values (Table 2, Line3, 4) of the DNA extracted by SDS method. To further verify that different cell lysates clearly affected the quality of the extracted DNA, two replicates of SDS and CTAB lysates were set up, and the results are shown in fig. 3, with different cell lysates significantly affecting the quality of the extracted DNA pairs (fig. 3). Finally, the effect of the cell collection method on the DNA purity was investigated, and the results showed that the genomic DNA obtained by extracting DNA from the algal cells collected by the filtration membrane method was of higher quality (FIG. 4, bands 5 and 6).

And (4) analyzing results:

the extraction of nucleic acid is the basis of the genetic and molecular biology experiments of marine microalgae. For specific harmful algae such as red tide bent algae, the traditional nucleic acid extraction method cannot obtain high-quality nucleic acid, and is not favorable for better and comprehensively understanding the biological characteristics of the red tide bent algae from the molecular level. Therefore, a rapid, simple and reliable nucleic acid extraction method is very necessary.

Find out the proper red tide heterocurvula cell cracking method and ideal cracking buffer solution. Selecting SDS lysate and CTAB lysate to respectively lyse algae cells under the incubation condition of 65 ℃. High concentrations of DNA were obtained using the SDS method (FIG. 2, Lane1), but the ratio of values for 260/280 and 260/230 was 1.66 and 0.94, respectively (Table 2, Lane1), indicating protein and phenol contamination, which affected the efficiency of downstream experiments. It is noted that, although the purity of the obtained DNA was improved by purifying the DNA using a DNA purification column after the extraction of the DNA using the SDS method (Table 1, Lane2), the process was time-consuming, the yield of the DNA was greatly reduced, and the integrity was low. The SDS lysate is not a proper lysate for extracting high-quality DNA of the red tide bent algae.

The quality of DNA extracted by a CTAB method and an SDS method is compared, and the different lysates have obvious influence on the quality of the red tide heterocurvula DNA. From fig. 3 and table 2 it can be derived: when DNA was extracted by the CTAB method, protein contamination was greatly reduced (FIG. 3, CTAB method:

bands

1, 2; SDS method: bands 3, 4). In Table 2, the DNA extracted by the SDS method and the CTAB method is shown to have better quality than the DNA extracted by the SDS method in comparison with 260/280 and 160/230.

Further verifies the influence of the cell collection method on the DNA extraction result. As a result, as shown in FIG. 4, the collection of algal cells using the filtration membrane resulted in obtaining a high concentration of DNA. While breaking cells using porcelain beads after the algal cells are collected by centrifugation will break the DNA into small fragments, affecting the integrity of the DNA and the quality of the DNA (FIG. 4, lane 2). This is because the red tide yizhuan algae is a kind of microalgae without cell wall, and is surrounded by the periplasmic membrane, and the centrifugation will make the cells gather together to form a cell mass, which will prevent the lysate from fully contacting the cells. Therefore, the collection of cells by centrifugation deteriorates the quality of DNA extraction. The method of using ceramic beads to disperse cells after centrifugal collection can disperse cell clusters and damage the integrity of DNA. In contrast, the use of a filter membrane to collect algal cells can effectively avoid the above-mentioned problems. However, a small amount of RNA could still be detected in the extraction results (FIG. 4,

bands

4, 5; Table 2, Lane4, 5), RNA contamination could be removed by the addition of RNAse without affecting the yield and integrity of DNA (FIG. 4,

bands

5, 6; Lane5, 6).

In conclusion, the optimized method can obtain higher-quality DNA, and the CTAB method is a proper cell lysate for obtaining high-quality DNA. However, to further improve the quality of DNA, the algal cells can be collected by a membrane filtration method, which not only has high yield, but also has good integrity, high purity, and guaranteed quality and quantity. In addition, the isolated genomic DNA is suitable for downstream processes such as PCR and high-throughput sequencing.

TABLE 1 Absorbance values of DNAs extracted by different methods

(FIG. 2, lane 1: DNA extraction by SDS method; lane 2: DNA purification by Zymo purification column after DNA extraction by SDS method; lane 3: DNA extraction by conventional method; M: DNAmarker).

TABLE 2 Absorbance values of DNAs extracted by different methods

(FIG. 3 shows

bands

1 and 2 showing DNA extracted by CTAB method;

bands

3 and 4 showing DNA extracted by SDS method; M shows DNAmarker molecular weight as in FIG. 2)

TABLE 3 Absorbance values of DNAs extracted by different methods

(FIG. 4 shows a band 1 in which algal cells were collected by a filtration method, a band 2 in which algal cells were collected by a centrifugation method and disrupted by ceramic beads,

bands

3 and 4 in which algal cells were collected by a filter and then disrupted by CTAB buffer solution,

bands

5 and 6 in which RNase was added in the same manner as in

bands

3 and 4, and M in which the molecular weight of DNAmarker is the same as in FIG. 2).

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention.

Claims

1. A method for extracting high-quality DNA from heterocurvulus akashiwo cells is characterized by comprising the following steps:

s1 culture of algae cells: filtering seawater for preparing algae culture solution with 0.22 μm filter membrane, sterilizing at high temperature and high pressure, and salinity of 30 PSU;

s2 screening of algal cell lysis buffer: obtaining high-quality DNA by an SDS treatment method and a CTAB extraction method; firstly, obtaining algae cells from a culture solution, and then suspending cell particles in SDS and CTAB cell lysis solutions respectively;

s3, collecting algae cells: using a 0.3-micron PVDF filter membrane, transferring the filter membrane into 0.8mL CTAB buffer solution preheated at 60 ℃, shearing the filter membrane by using scissors, sealing the opening of a centrifuge tube by using a sealing membrane, incubating at 60 ℃ for 2 hours, slightly inverting and mixing from time to time, centrifuging the sample at 12000rpm for 15s, and transferring cell lysate into a new centrifuge tube;

s5, transferring the upper aqueous phase into a new centrifuge tube, adding RNase, and carrying out warm bath at 37 ℃ for 30 minutes;

2. The method of claim 1, wherein the DNA is extracted from Heterocurus akashiwo cells in high quality: the culture medium for culturing the algae cells in the S1 is f/2 culture medium without adding silicon element, the algae cells are cultured under the culture condition of 20 ℃, the concentration of the algae cells is counted under a microscope by using a Sedgwick-Rafter counting plate, and then the concentration of the cells is calculated.

3. The method of claim 2, wherein the DNA is extracted from the Heterocurus akashiwo cells in high quality: the calculation formula of the algae cell concentration is as follows: c_ADR G50, wherein C_AIndicating the concentration of the algae cells; DR represents the dilution factor of the algae culture solution during the algae cultivation; g represents the average value of the number of algal cells.

4. The method of claim 1, wherein the DNA is extracted from Heterocurus akashiwo cells in high quality: the specific steps of lysis of algal cells by SDS in S2 are as follows:

s2-1-1: collecting algae cells by centrifugation, suspending cell particles in SDS lysate (0.1M EDTA, 1% SDS), and incubating the cell suspension at 56 ℃ overnight;

s2-1-2: an equal volume of chloroform was added to the cell lysate and mixed by gentle inversion. Centrifuging at 12000rpm for 10 minutes at 4 deg.C, carefully transferring the supernatant to a new centrifuge tube, adding 1/10 volumes of NaAc and 2/3 volumes of isopropanol;

s2-1-3: the mixed cell lysate is stood for 10 minutes at room temperature, centrifuged for 5 minutes at 4 ℃ and 12000rpm, DNA is collected, washed twice with 70% absolute ethyl alcohol and dried, and then the DNA is dissolved in sterile water.

5. The method of claim 1, wherein the DNA is extracted from Heterocurus akashiwo cells in high quality: the specific steps of disrupting algal cells using CTAB in S2 are as follows:

s2-2-1: incubation in 0.5ml DNA lysis buffer (10mM Tris, pH 8.0; 100mM EDTA, pH 8.0; 0.5% SDS and 200. mu.g/ml proteinase K) at 55 ℃ for 5 hours; add 82.5. mu.L of 5M NaCl to a final concentration of 0.7M;

s2-2-2: then adding 82.5 μ L of pre-preheated 10% (w/v) CTAB solution to the mixture, vortexing for 1 minute, and incubating at 56 deg.C for 10 minutes;

s2-2-3: add 600. mu.L of chloroform at 12000rpm, centrifuge for 10 minutes, transfer the upper aqueous phase to a new centrifuge tube, and add 2 volumes of DNA buffer to each DNA sample, as in Zymo's DNA purification and concentration kit.

6. The method of claim 1, wherein the DNA is extracted from Heterocurus akashiwo cells in high quality: the method for determining the DNA concentration in S9: according to the absorbance values at 230, 260 and 280nm, the purity of the nucleic acid is quantitatively determined by using Nanodrop, the ratio of 260/280 to 260/230 is calculated, the optimized method is used for extracting the genome DNA of the Heterocurus akashiwo, the integrity of the genome DNA is identified by 0.6% agarose gel electrophoresis, and the DNA bands are observed by using a gel nucleic acid staining method.