CN111607636A - A method for extracting high-quality DNA from cells of Heteroflexus alba - Google Patents

A method for extracting high-quality DNA from cells of Heteroflexus alba Download PDF

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CN111607636A
CN111607636A CN202010597957.4A CN202010597957A CN111607636A CN 111607636 A CN111607636 A CN 111607636A CN 202010597957 A CN202010597957 A CN 202010597957A CN 111607636 A CN111607636 A CN 111607636A
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胡广伟
张珍珍
姬南京
申欣
蔡月凤
覃昊
石一彪
邓圣贤
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Jiangsu Ocean University
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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

一种从赤潮异弯藻细胞中提取高质量DNA的方法A method for extracting high-quality DNA from cells of Heteroflexus alba

技术领域technical field

本发明涉及赤潮异弯藻DNA提取领域,具体为一种从赤潮异弯藻细胞中提取高质量DNA的方法。The invention relates to the field of DNA extraction of Heteroflexus alba, in particular to a method for extracting high-quality DNA from cells of Heteroflexus alba.

背景技术Background technique

有害藻华是由于某些藻类爆发性增殖而形成的,可对海洋生态系统和当地经济造成重大破坏。近年来,有害藻华的发生频率、强度和地理分布在世界各地都有所增加。赤潮异弯藻是典型的赤潮物种,是一种广温、广盐性生物,分布在世界多个海域。这种藻之所以被广泛研究,因为它在赤潮形成时会产生鱼毒素,严重破坏海洋生态系统,导致大量人工养殖鱼类死亡。然而,关于藻华形成和毒素产生机制仍不清楚,为了更好地了解赤潮异弯藻如何形成藻华、形成藻华时如何杀死鱼类,有关分子机制或调节该物种生长和新陈代谢的生化过程的信息是非常重要的。Harmful algal blooms form as a result of certain algal blooms that can cause significant damage to marine ecosystems and local economies. The frequency, intensity and geographic distribution of HABs have increased around the world in recent years. Heterocurbita albicans is a typical red tide species, a ubiquitous and basal organism, distributed in many sea areas of the world. This algae has been widely studied because it produces fish toxins when red tides form, severely damaging marine ecosystems and killing large numbers of farmed fish. However, the mechanisms of algal bloom formation and toxin production remain unclear, to better understand how H. algal blooms form, how algal blooms kill fish, the molecular mechanisms or the biochemistry that regulates the growth and metabolism of this species Process information is very important.

基于DNA的分子生物学方法是研究生物特征的有效方法之一。对于分子分析、基因工程,如克隆和DNA测序,所有这些步骤的初始步骤都是DNA提取。目前有很多种DNA提取方法,如十二烷基硫酸钠(SDS)法,十六烷基三甲基溴化铵(CTAB)法和商业化的DNA提取试剂盒。然而,上述的一些DNA提取方法可能适用于某些特定的生物,但不适用于特定的藻类物种。赤潮异弯藻的基因组较大,很难获得完整的基因组DNA,因此,得到高浓度、高纯度和高完整度的赤潮异弯藻基因组是开展该物种DNA高通量测序技术的先决条件。DNA-based molecular biology methods are one of the effective methods to study biological characteristics. For molecular analysis, genetic engineering, such as cloning and DNA sequencing, the initial step in all of these steps is DNA extraction. There are many DNA extraction methods, such as sodium dodecyl sulfate (SDS) method, cetyltrimethylammonium bromide (CTAB) method and commercial DNA extraction kits. However, some of the DNA extraction methods described above may work for some specific organisms but not for specific algal species. The genome of H. alba is large, and it is difficult to obtain complete genomic DNA. Therefore, obtaining a high-concentration, high-purity and high-integrity genome of H. alba is a prerequisite for high-throughput DNA sequencing technology of this species.

从植物和藻类中提取DNA的困难之处在于它们具有独特的细胞结构。赤潮异弯藻的细胞很脆弱,没有细胞壁。所以在用离心法收集藻细胞时,细胞会聚集形成细胞团,使藻细胞的完全裂解变得更加困难。虽然匀浆器可以破坏细胞的结构,但是物理操作会降低基因组DNA的完整性,使得这种方法获得的DNA不适合于基因组测序等下游实验。The difficulty in extracting DNA from plants and algae is that they have unique cellular structures. The cells of Heteroflexus alba are fragile and have no cell walls. Therefore, when algal cells are collected by centrifugation, the cells aggregate to form cell clusters, making complete lysis of the algal cells more difficult. Although homogenizers can disrupt the structure of cells, physical manipulation can reduce the integrity of genomic DNA, making the DNA obtained by this method unsuitable for downstream experiments such as genome sequencing.

发明内容SUMMARY OF THE INVENTION

本发明的目的是针对现有技术的缺陷,提出了一种从赤潮异弯藻细胞中提取高质量DNA的方法,以解决上述背景技术提出的问题。The purpose of the present invention is to address the defects of the prior art, and propose a method for extracting high-quality DNA from cells of Heteroflexus alba, so as to solve the problems raised by the above-mentioned background technology.

为实现上述目的,本发明提供如下技术方案:一种从赤潮异弯藻细胞中提取高质量DNA的方法,包括如下步骤:To achieve the above object, the present invention provides the following technical solutions: a method for extracting high-quality DNA from Heteroflexus alba cells, comprising the following steps:

S1:藻细胞的培养:用于配制藻培养液的海水经0.22μm的滤膜过滤,并经高压灭菌,盐度为30PSU;S1: Cultivation of algal cells: the seawater used to prepare the algal culture solution is filtered through a 0.22 μm filter membrane, and sterilized by autoclaving, with a salinity of 30 PSU;

S2:藻细胞裂解缓冲液的筛选:采用SDS处理法和CTAB提取法获得高质量的DNA;首先从培养基中获取藻细胞,然后将细胞颗粒分别悬浮在SDS和CTAB细胞裂解液中;S2: Screening of algal cell lysis buffer: high-quality DNA was obtained by SDS treatment and CTAB extraction; first, algal cells were obtained from the medium, and then the cell pellets were suspended in SDS and CTAB cell lysates, respectively;

S3:收集藻细胞:使用0.3μm的PVDF过滤膜,然后将过滤膜转移到0.8mL于60℃预热的CTAB细胞裂解液(2%CTAB,100mMTris-HClpH8.0,1.4MNaCL,0.2%β巯基乙醇,0.1mg/ml蛋白酶K)中,用剪刀将过滤膜剪碎,用封口膜密封离心管管口,60℃孵育2小时,不时轻轻地颠倒混匀,样品在12000rpm离心15s后,将细胞裂解液转移到新的离心管中;S3: Harvest algal cells: use a 0.3 μm PVDF filter membrane, then transfer the filter membrane to 0.8 mL of CTAB cell lysate (2% CTAB, 100 mM Tris-HCl pH 8.0, 1.4 M NaCl, 0.2% β-thiol) pre-warmed at 60 °C ethanol, 0.1 mg/ml proteinase K), cut the filter membrane into pieces with scissors, seal the mouth of the centrifuge tube with parafilm, incubate at 60°C for 2 hours, and gently invert and mix occasionally. Transfer the cell lysate to a new centrifuge tube;

S4:添加800μL的氯仿至细胞液中,轻轻地颠倒离心管2分钟使其混匀,在室温下静置2分钟后,4℃,12000rpm离心10分钟;S4: Add 800 μL of chloroform to the cell solution, invert the centrifuge tube gently for 2 minutes to mix it, stand at room temperature for 2 minutes, and then centrifuge at 4°C and 12000rpm for 10 minutes;

S5:转移上层水相至新的离心管中,添加RNA酶,37℃温育30分钟;S5: transfer the upper aqueous phase to a new centrifuge tube, add RNase, and incubate at 37°C for 30 minutes;

S6:加入等体积的氯仿至温浴后的混合液中,12000rpm离心10分钟;S6: add equal volume of chloroform to the mixed solution after the warm bath, and centrifuge at 12,000 rpm for 10 minutes;

S7:将上层水相转移到另一个新的离心管中,添加1/10体积的醋酸钠和2/3体积的异丙醇,轻轻颠倒混匀;S7: transfer the upper aqueous phase to another new centrifuge tube, add 1/10 volume of sodium acetate and 2/3 volume of isopropanol, gently invert and mix;

S8:室温下,静置10分钟,4℃,12000rpm,离心5分钟。去掉上清,用70%的无水乙醇洗涤2次,吸掉无水乙醇,室温晾干;最后,将干燥后的DNA样品重新悬浮于100μL无菌水中,用0.6%的琼脂糖凝胶进行电泳;S8: at room temperature, stand for 10 minutes, 4° C., 12000 rpm, and centrifuge for 5 minutes. Remove the supernatant, wash twice with 70% anhydrous ethanol, aspirate the anhydrous ethanol, and air dry at room temperature; finally, resuspend the dried DNA sample in 100 μL sterile water, and use 0.6% agarose gel for electrophoresis;

S9:DNA浓度、纯度和完整性的分析:采用超微量分光光度计定量测定DNA浓度。S9: Analysis of DNA Concentration, Purity and Integrity: DNA concentration was quantitatively determined using an ultra-micro spectrophotometer.

作为本发明的一种优选技术方案,所述S1中的培养藻细胞的培养基是不添加硅元素的f/2培养基,在20℃培养条件下培养,藻细胞浓度是使用Sedgwick-Rafter计数板在显微镜下进行细胞计数,后计算细胞浓度。As a preferred technical solution of the present invention, the medium for culturing algal cells in the S1 is f/2 medium without adding silicon element, cultured at 20°C, and the algal cell concentration is counted using Sedgwick-Rafter Plates were counted under a microscope and cell concentrations were calculated.

作为本发明的一种优选技术方案,所述藻细胞浓度的计算公式为:CA=DR*G*50,其中CA表示藻细胞浓度;DR表示数藻时藻培养液的稀释倍数;G表示藻细胞数的平均值。As a preferred technical solution of the present invention, the calculation formula of the algal cell concentration is: CA = DR*G*50, wherein CA represents the algal cell concentration; DR represents the dilution ratio of the algal culture solution when counting algae; G Indicates the average number of algal cells.

作为本发明的一种优选技术方案,所述S2中使用SDS裂解藻细胞的具体步骤:As a preferred technical solution of the present invention, the specific steps of using SDS to lyse algal cells in the S2:

S2-1-1:离心收集藻细胞,细胞颗粒悬浮于SDS细胞裂解液(0.1MEDTA,1%SDS)中,细胞悬液于56℃孵育过夜;S2-1-1: algal cells were collected by centrifugation, the cell pellets were suspended in SDS cell lysate (0.1 MEDTA, 1% SDS), and the cell suspension was incubated at 56°C overnight;

S2-1-2:将等体积的氯仿加入到细胞悬液中,反复颠倒混匀,于4℃,12000rpm,离心10分钟,小心地将上层水相转移到新的离心管中,加入1/10体积的NaAc和2/3体积的异丙醇;S2-1-2: Add an equal volume of chloroform to the cell suspension, invert and mix repeatedly, centrifuge at 4°C, 12,000 rpm for 10 minutes, carefully transfer the upper aqueous phase to a new centrifuge tube, add 1/ 10 volumes of NaAc and 2/3 volumes of isopropanol;

S2-1-3:将混匀的细胞悬液在室温下静置10分钟后于4℃,12000rpm,离心5分钟,收集DNA,用70%的无水乙醇洗涤DNA两次后晾干,后在无菌水中溶解DNA。S2-1-3: The mixed cell suspension was allowed to stand at room temperature for 10 minutes, then centrifuged at 4°C, 12,000 rpm for 5 minutes to collect DNA. The DNA was washed twice with 70% absolute ethanol and then air-dried. Dissolve DNA in sterile water.

作为本发明的一种优选技术方案,所述S2中使用CTAB裂解藻细胞的具体步骤:As a preferred technical solution of the present invention, the concrete steps of using CTAB to lyse algal cells in the S2:

S2-2-1:在0.5mlDNA裂解缓冲液(10mMTris,pH8.0;100mMEDTA,pH8.0;0.5%SDS和200μg/ml的蛋白酶K)中于55℃孵育5小时;添加82.5μL5MNaCl,终浓度为0.7M;S2-2-1: Incubate in 0.5 ml DNA lysis buffer (10 mM Tris, pH 8.0; 100 mM EDTA, pH 8.0; 0.5% SDS and 200 μg/ml proteinase K) for 5 hours at 55°C; add 82.5 μL 5M NaCl, final concentration is 0.7M;

S2-2-2:然后添加82.5μL提前预热的10%(w/v)CTAB溶液至细胞裂解液中,涡旋1分钟后在56℃条件下温育10分钟;S2-2-2: Then add 82.5 μL of pre-warmed 10% (w/v) CTAB solution to the cell lysate, vortex for 1 minute and incubate at 56°C for 10 minutes;

S2-2-3:添加600μL的氯仿,12000rpm,离心10分钟,将上清液转移到新的离心管中,然后在每个DNA样品中加入2倍体积的DNA结合缓冲液,其余步骤与Zymo的DNA纯化和浓缩试剂盒方法相同。S2-2-3: Add 600 μL of chloroform, 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, the rest of the steps are the same as Zymo The DNA purification and concentration kit methods are the same.

作为本发明的一种优选技术方案,所述S9中测定DNA浓度的方法:根据在230、260和280nm处的吸光度值,用Nanodrop定量测定核酸的纯度,并计算260/280和260/230的比值,用优化后的方法提取基因组DNA,经0.6%琼脂糖凝胶电泳鉴定其完整性,用凝胶核酸染色法观察DNA条带。As a preferred technical solution of the present invention, the method for determining DNA concentration in S9: according to the absorbance values at 230, 260 and 280 nm, use Nanodrop to quantitatively determine the purity of nucleic acid, and calculate the 260/280 and 260/230 The genomic DNA was extracted by the optimized method, its integrity was identified by 0.6% agarose gel electrophoresis, and the DNA bands were observed by gel nucleic acid staining.

本发明的有益效果:本发明所提出的一种从赤潮异弯藻细胞中提取高质量DNA的方法,主要通过使用过滤膜收集赤潮异弯藻细胞的方法和选择合适的CTAB细胞裂解液。结果表明:新方法在提取得到的赤潮异弯藻的基因组DNA的浓度和质量上均优于其他方法,适用于PCR分析和DNA高通量测序。Beneficial effects of the present invention: The method for extracting high-quality DNA from Heteroflexus alba cells proposed by the present invention mainly uses a filter membrane to collect Heteroflexus alba cells and selects a suitable CTAB cell lysate. The results showed that the new method was superior to other methods in the concentration and quality of the extracted genomic DNA of Heteroflexus alba, and was suitable for PCR analysis and high-throughput DNA sequencing.

附图说明Description of drawings

图1是本发明的流程图;Fig. 1 is the flow chart of the present invention;

图2标准电泳法分析使用不同的方法提取得到的基因组DNA的完整性和产量;Figure 2. Analysis of the integrity and yield of genomic DNA extracted using different methods by standard electrophoresis;

图3标准电泳法鉴定DNA完整性;Figure 3 identifies DNA integrity by standard electrophoresis;

图4用标准电泳法评价DNA质量。Figure 4 Assessment of DNA quality by standard electrophoresis.

具体实施方式Detailed ways

下面对本发明的较佳实施例进行详细阐述,以使本发明的优点和特征能更易被本领域人员理解,从而对本发明的保护范围做出更为清楚明确的界定。The preferred embodiments of the present invention are described in detail below, so that the advantages and features of the present invention can be more easily understood by those skilled in the art, and the protection scope of the present invention can be more clearly defined.

实施例1:请参阅图1,本发明提供一种技术方案:一种从赤潮异弯藻细胞中提取高质量DNA的方法:本发明的实现步骤如下:Embodiment 1: Please refer to FIG. 1, the present invention provides a technical solution: a method for extracting high-quality DNA from Heteroflexus alba cells: The implementation steps of the present invention are as follows:

包括如下步骤:It includes the following steps:

S1:藻细胞的培养:用于配制藻培养液的海水经0.22μm的滤膜过滤,并经高压灭菌,盐度为30PSU;S1: Cultivation of algal cells: the seawater used to prepare the algal culture solution is filtered through a 0.22 μm filter membrane, and sterilized by autoclaving, with a salinity of 30 PSU;

培养藻细胞的培养基是不添加硅元素的f/2培养基,在20℃培养条件下培养,藻细胞浓度是使用Sedgwick-Rafter计数板在显微镜下进行细胞计数,后计算细胞浓度。The medium for culturing algal cells was f/2 medium without added silicon element, cultured at 20°C, and the algal cell concentration was counted under a microscope using a Sedgwick-Rafter counting plate, and then the cell concentration was calculated.

所述藻细胞浓度的计算公式为:CA=DR*G*50,其中CA表示藻细胞浓度;DR表示数藻时藻培养液的稀释倍数;G表示藻细胞数的平均值。The calculation formula of the algal cell concentration is: CA =DR*G*50, wherein CA represents the algal cell concentration; DR represents the dilution ratio of the algal culture solution when counting algae; G represents the average value of the algal cell number.

S2:藻细胞裂解缓冲液的筛选:采用SDS处理法和CTAB提取法获得高质量的DNA;首先从培养基中获取藻细胞,然后将细胞颗粒分别悬浮在SDS和CTAB细胞裂解液中;S2: Screening of algal cell lysis buffer: high-quality DNA was obtained by SDS treatment and CTAB extraction; first, algal cells were obtained from the medium, and then the cell pellets were suspended in SDS and CTAB cell lysates, respectively;

使用SDS裂解藻细胞的具体步骤:Specific steps for lysing algal cells using SDS:

S2-1-1:离心收集藻细胞,细胞颗粒悬浮于SDS缓冲液(0.1MEDTA,1%SDS)中,细胞悬液于56℃孵育过夜;S2-1-1: algal cells were collected by centrifugation, the cell pellets were suspended in SDS buffer (0.1 MEDTA, 1% SDS), and the cell suspension was incubated at 56°C overnight;

S2-1-2:将等体积的氯仿加入到细胞裂解液中,轻轻地颠倒混匀,于4℃,12000rpm,离心10分钟。小心地将上层水相转移到新的离心管中,加入1/10体积的NaAc和2/3体积的异丙醇;S2-1-2: Add an equal volume of chloroform to the cell lysate, gently invert and mix, and centrifuge at 4°C, 12000 rpm for 10 minutes. Carefully transfer the upper aqueous phase to a new centrifuge tube, add 1/10 volume NaAc and 2/3 volume isopropanol;

S2-1-3:将混匀的细胞裂解液在室温下静置10分钟后于4℃,12000rpm,离心5分钟,收集DNA。用70%的无水乙醇洗涤DNA两次后晾干,后在无菌水中溶解DNA。S2-1-3: The mixed cell lysate was allowed to stand for 10 minutes at room temperature, and then centrifuged at 4°C and 12000 rpm for 5 minutes to collect DNA. The DNA was washed twice with 70% absolute ethanol and air-dried before dissolving the DNA in sterile water.

使用CTAB裂解藻细胞的具体步骤:Specific steps for lysing algal cells with CTAB:

S2-2-1:在0.5mlDNA裂解缓冲液(10mMTris,pH8.0;100mMEDTA,pH8.0;0.5%SDS和200μg/ml的蛋白酶K)中于55℃孵育5小时;添加82.5μL5MNaCl,终浓度为0.7M;S2-2-1: Incubate in 0.5 ml DNA lysis buffer (10 mM Tris, pH 8.0; 100 mM EDTA, pH 8.0; 0.5% SDS and 200 μg/ml proteinase K) for 5 hours at 55°C; add 82.5 μL 5M NaCl, final concentration is 0.7M;

S2-2-2:然后添加82.5μL提前预热的10%(w/v)CTAB溶液至裂解液中,涡旋1分钟后在56℃条件下温浴10分钟;S2-2-2: Then add 82.5 μL of pre-warmed 10% (w/v) CTAB solution to the lysate, vortex for 1 minute, and incubate at 56°C for 10 minutes;

S2-2-3:添加600μL的氯仿,12000rpm,离心10分钟,将上层水相转移到新的离心管中,然后在每个DNA样品中加入2倍体积的DNA结合缓冲液,其余步骤与Zymo的DNA纯化和浓缩试剂盒方法相同。S2-2-3: Add 600 μL of chloroform, 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 steps are the same as Zymo The DNA purification and concentration kit methods are the same.

S3:收集藻细胞:使用0.3μm的PVDF过滤膜,然后将过滤膜转移到0.8mL于60℃预热的CTAB缓冲液(2%CTAB,100mMTris-HClpH8.0,1.4MNaCL,0.2%β巯基乙醇,0.1mg/ml蛋白酶K)中,用剪刀将过滤膜剪碎,用封口膜密封瓶盖,60℃孵育2小时,不时地轻轻颠倒混匀,样品在12000rpm离心15s后,将细胞裂解液转移到新的离心管中;S3: Harvest algal cells: use a 0.3 μm PVDF filter, then transfer the filter to 0.8 mL of CTAB buffer (2% CTAB, 100 mM Tris-HCl pH 8.0, 1.4 M NaCl, 0.2% β-mercaptoethanol) pre-warmed at 60°C , 0.1mg/ml proteinase K), cut the filter membrane with scissors, seal the bottle cap with parafilm, incubate at 60°C for 2 hours, gently invert and mix occasionally, after centrifuging the sample at 12000rpm for 15s, remove the cell lysate Transfer to a new centrifuge tube;

S4:添加800μL的氯仿至细胞裂解液中,轻轻地颠倒离心管2分钟使其混匀,在室温下静置2分钟后,4℃,12000rpm离心10分钟;S4: Add 800 μL of chloroform to the cell lysate, invert the centrifuge tube gently for 2 minutes to mix it, let it stand for 2 minutes at room temperature, and then centrifuge at 4°C and 12,000 rpm for 10 minutes;

S5:转移上层水相转移至新的离心管中,添加RNA酶,37℃温浴30分钟;S5: transfer the upper aqueous phase to a new centrifuge tube, add RNase, and incubate at 37°C for 30 minutes;

S6:加入等体积的氯仿至温浴后的细胞裂解液中,12000rpm离心10分钟;S6: add an equal volume of chloroform to the cell lysate after the warm bath, and centrifuge at 12,000 rpm for 10 minutes;

S7:将上层水相转移到另一个新的离心管中,添加1/10体积的醋酸钠和2/3体积的异丙醇,轻轻地颠倒混匀;S7: transfer the upper aqueous phase to another new centrifuge tube, add 1/10 volume of sodium acetate and 2/3 volume of isopropanol, and gently invert and mix;

S8:室温下,静置10分钟,4℃,12000rpm,离心5分钟,去掉上清。用70%的无水乙醇洗涤沉淀2次,吸掉无水乙醇,室温晾干;最后,将干燥后的DNA样品重新悬浮于100μL无菌水中,用0.6%的琼脂糖凝胶进行电泳;S8: at room temperature, let stand for 10 minutes, 4° C., 12000 rpm, centrifuge for 5 minutes, and remove the supernatant. Wash the precipitate twice with 70% absolute ethanol, remove the absolute ethanol, and dry at room temperature; finally, resuspend the dried DNA sample in 100 μL of sterile water, and perform electrophoresis on 0.6% agarose gel;

S9:DNA浓度、纯度和完整性的分析:使用超微量分光光度计定量测定DNA浓度。根据在230、260和280nm处的吸光度值,用Nanodrop定量测定核酸的纯度,并计算260/280和260/230的值,用优化后的方法提取赤潮异弯藻的基因组DNA,经0.6%琼脂糖凝胶电泳鉴定其完整性,用凝胶核酸染色法观察DNA条带。S9: Analysis of DNA Concentration, Purity, and Integrity: DNA concentration was quantified using an ultra-micro spectrophotometer. According to the absorbance values at 230, 260 and 280 nm, the purity of nucleic acid was quantitatively determined with Nanodrop, and the values of 260/280 and 260/230 were calculated. The optimized method was used to extract the genomic DNA of Heteroflexus alba, which was purified by 0.6% agar. Its integrity was identified by glycogel electrophoresis, and DNA bands were observed by gel nucleic acid staining.

结果1:DNA的完整度Outcome 1: Integrity of DNA

如图2所示,用SDS法、SDS法提取DNA后用洗脱柱进行DNA纯化和实验室常用的常规提取DNA的方法分别获得赤潮异弯藻的基因组DNA,对其凝胶电泳结果进行分析。利用SDS法从赤潮异弯藻中提取得到的DNA显示出一个强条带(图2,条带1)。当使用SDS法提取DNA后用Zymo纯化试剂盒进行纯化后,发现条带很弱(图2,条带2)。琼脂糖凝胶电泳结果显示使用标准的DNA提取方法得到的DNA在电泳后条带弥散(图2,条带3)。通过分析这三种方法提取得到的赤潮异弯藻DNA的260/280、260/230的值以及得到的DNA的完整度,发现得到的DNA纯度不高或完整度较低。此外,藻细胞的收取方法也影响DNA的浓度,用滤膜法收集藻细胞呈现出很强的条带(图4,条带1),说明提取得到的DNA浓度很高,而使用磁珠对离心收集的细胞进行破碎,提取得到的DNA的电泳结果显示DNA条带弥散(图4,条带2),说明提取得到的DNA完整度低。比较了CTAB法和SDS法提取的DNA的质量,表明用CTAB法进行DNA提取可以减少蛋白质的污染,说明CTAB法更适合于提取赤潮异弯藻的基因组DNA。As shown in Figure 2, the genomic DNA of Heteroflexus akataide was obtained by SDS method and SDS method, followed by DNA purification with elution column and the routine DNA extraction method commonly used in laboratories, and the results of gel electrophoresis were analyzed. . DNA extracted from Heteroflexus alba by SDS method showed a strong band (Fig. 2, band 1). When DNA was extracted by SDS method and purified with Zymo purification kit, a very weak band was found (Figure 2, band 2). The results of agarose gel electrophoresis showed that DNA obtained using standard DNA extraction methods had a diffuse band after electrophoresis (FIG. 2, band 3). By analyzing the values of 260/280, 260/230 and the integrity of the DNA extracted by the three methods, it was found that the purity of the DNA was not high or the integrity was low. In addition, the collection method of algal cells also affects the concentration of DNA. The algal cells collected by the filter method showed a strong band (Fig. 4, band 1), indicating that the concentration of extracted DNA was high, and the use of magnetic beads to The cells collected by centrifugation were disrupted, and the electrophoresis results of the extracted DNA showed that the DNA bands were dispersed (Fig. 4, band 2), indicating that the integrity of the extracted DNA was low. The quality of DNA extracted by CTAB method and SDS method was compared, and it was shown that DNA extraction by CTAB method could reduce the pollution of protein.

结果2:DNA的纯度Result 2: Purity of DNA

通过计算260/280和260/230的值来估算核酸纯度,260/280的值可以作为是否存在蛋白污染的指标,同样260/230的比值可以用来监测其他污染物,如碳水化合物、硫氰酸胍和酚类。SDS法提取DNA的260/280值为1.66,260/230的值为0.94(表1,Lane1);当用DNA纯化柱进一步纯化分离出的DNA时,260/280和260/230的比值分别提高到1.75和1.59(表1,Lane2)。相比而言,使用标准法提取得到的DNA的260/280和260/230的值分别为1.83和2.03(表1,Lane3)。相比于SDS法提取得到的DNA的260/280和260/230的(表2,Line3、4)值用CTAB法提取得到的DNA的260/280和260/230值较好(表2,Line1、2)。为了进一步验证不同的细胞裂解液明显会对提取的DNA的质量产生影响,SDS和CTAB裂解液设置两个重复,结果如图3所示,不同的细胞裂解液明显影响DNA对的提取质量(图3)。最后,研究了细胞收集方法对DNA纯度的影响,结果表明,用滤膜法收集的藻细胞进行DNA的提取,获得的基因组DNA的质量更高(图4,条带5和6)。The nucleic acid purity is estimated by calculating the values of 260/280 and 260/230. The value of 260/280 can be used as an indicator of whether there is protein contamination. Similarly, the ratio of 260/230 can be used to monitor other contaminants such as carbohydrates, thiocyanate Guanidine acids and phenols. The 260/280 value of DNA extracted by SDS method was 1.66, and the value of 260/230 was 0.94 (Table 1, Lane 1); when the isolated DNA was further purified with a DNA purification column, the ratios of 260/280 and 260/230 were increased, respectively to 1.75 and 1.59 (Table 1, Lane 2). In comparison, the 260/280 and 260/230 values for DNA extracted using the standard method were 1.83 and 2.03, respectively (Table 1, Lane 3). Compared with the 260/280 and 260/230 (Table 2, Line 3, 4) values of the DNA extracted by the SDS method, the 260/280 and 260/230 values of the DNA extracted by the CTAB method were better (Table 2, Line 1 ,2). In order to further verify that different cell lysates obviously affect the quality of the extracted DNA, two replicates were set for SDS and CTAB lysates, and the results are shown in Figure 3. 3). Finally, the effect of cell collection method on DNA purity was investigated, and the results showed that DNA extraction from algal cells collected by the filter method resulted in higher quality genomic DNA (Fig. 4, lanes 5 and 6).

结果分析:Result analysis:

核酸的提取是海洋微藻遗传和分子生物学实验的基础。对于赤潮异弯藻等特定的有害藻类,传统的核酸提取方法无法获得高质量的核酸,不利于从分子水平更好地全面了解赤潮异弯藻的生物学特性。因此,快速、简单、可靠的核酸提取方法非常必要。The extraction of nucleic acid is the basis of genetic and molecular biology experiments of marine microalgae. For specific harmful algae such as H. alba, traditional nucleic acid extraction methods cannot obtain high-quality nucleic acids, which is not conducive to a better and comprehensive understanding of the biological characteristics of H. alba at the molecular level. Therefore, a fast, simple and reliable nucleic acid extraction method is very necessary.

寻找合适的赤潮异弯藻细胞的裂解方法和理想的裂解缓冲液。选择SDS裂解液、CTAB裂解液分别在65℃孵育条件下裂解藻细胞。使用SDS法可以得到高浓度的DNA(图2,条带1),但260/280和260/230的值比分别为1.66和0.94(表2,Lane1),表明受到了蛋白质和苯酚污染,这将影响下游实验的效率。值得注意的是,虽然使用SDS法进行DNA提取后使用DNA纯化柱纯化DNA可以提高得到的DNA的纯度(表1,Lane2),但是这一过程非常耗时,会大大降低DNA的产量,且完整度低。说明SDS裂解液并不是提取赤潮异弯藻高质量DNA的合适裂解液。Find a suitable lysis method and ideal lysis buffer for H. akashio cells. Select SDS lysate and CTAB lysate to lyse algal cells under incubation conditions at 65°C. High concentrations of DNA were obtained using the SDS method (Figure 2, Lane 1), but the ratios of 260/280 and 260/230 values were 1.66 and 0.94, respectively (Table 2, Lane 1), indicating protein and phenol contamination, which will affect the efficiency of downstream experiments. It is worth noting that although using SDS method for DNA extraction followed by DNA purification using a DNA purification column can improve the purity of the obtained DNA (Table 1, Lane 2), this process is very time-consuming, will greatly reduce the yield of DNA, and the complete low degree. It shows that SDS lysate is not a suitable lysate for extracting high-quality DNA from Heteroflexus alba.

比较了使用CTAB法和SDS法提取得到的DNA的质量,发现不同的裂解液对赤潮异弯藻DNA的质量有明显影响。根据图3和表2可以得出:当使用CTAB法提取DNA时,可以大大减少蛋白质污染(图3,CTAB法:条带1、2;SDS法:条带3、4)。表2中SDS法和CTAB法提取得到的DNA的260/280和160/230相比,表明CTAB法比SDS法提取的DNA质量更好。The quality of DNA extracted by CTAB method and SDS method was compared, and it was found that different lysates had obvious influence on the quality of DNA of Heteroflexus alba. According to Figure 3 and Table 2, it can be concluded that when DNA is extracted by CTAB method, protein contamination can be greatly reduced (Figure 3, CTAB method: bands 1 and 2; SDS method: bands 3 and 4). The comparison of 260/280 and 160/230 of DNA extracted by SDS method and CTAB method in Table 2 shows that the quality of DNA extracted by CTAB method is better than that of SDS method.

进一步验证了细胞收集方法对DNA提取结果的影响。结果如图4所示,使用过滤膜收集藻细胞会获得高浓度的DNA。而藻细胞通过离心方法收集后使用瓷珠打破细胞,会将DNA打破成小片段,影响DNA的完整性和DNA的质量(图4,条带2)。这是因为赤潮异弯藻是一种无细胞壁的微藻,被周质膜包围,离心将使细胞聚集在一起,形成一个细胞团,将阻碍裂解液充分接触细胞。因此,离心收集细胞会降低DNA的提取质量。离心收集后使用瓷珠打散细胞的办法可以使细胞团被打散,同时也会破坏DNA的完整性。相比之下,使用滤膜收集藻类细胞可以有效地避免上述问题。但是提取结果中仍能检测到少量RNA(图4,条带4、5;表2,Lane4、5),可以添加RNA酶去除RNA污染且对DNA的产量和完整性无影响(图4,条带5、6;Lane5、6)。The effect of cell collection method on DNA extraction results was further verified. The results are shown in Figure 4. The use of filter membranes to collect algal cells resulted in high concentrations of DNA. The algal cells were collected by centrifugation and then broken with ceramic beads, which would break the DNA into small fragments, affecting the integrity and quality of the DNA (Figure 4, band 2). This is because H. akata is a microalgae without a cell wall, surrounded by a periplasmic membrane, and centrifugation will cause the cells to clump together into a cell mass that will prevent the lysate from fully contacting the cells. Therefore, harvesting cells by centrifugation can reduce the quality of DNA extraction. The method of using ceramic beads to break up the cells after centrifugation can break up the cell mass and destroy the integrity of the DNA. In contrast, the use of filters to collect algal cells can effectively avoid the above problems. However, a small amount of RNA could still be detected in the extraction results (Figure 4, Lanes 4 and 5; Table 2, Lanes 4 and 5), and RNase could be added to remove RNA contamination without affecting the yield and integrity of DNA (Figure 4, Lanes 4 and 5). Belt 5, 6; Lane 5, 6).

综上所述,我们优化后的方法可以获得更高质量的DNA,CTAB法是获得高质量DNA的合适细胞裂解液。然而,为了进一步提高DNA质量,可以使用滤膜法收集藻细胞,该方法不仅产率高,而且完整性好,纯度高,质量和数量都得到保证。此外,分离出的基因组DNA适用于PCR和高通量测序等下游过程。In conclusion, our optimized method can obtain higher quality DNA, and CTAB method is a suitable cell lysate for obtaining high quality DNA. However, to further improve DNA quality, algal cells can be collected using a filter method, which not only has high yields, 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.

Figure BDA0002557723370000091
Figure BDA0002557723370000091

表1使用不同方法提取得到的DNA的吸光值Table 1 Absorbance values of DNA extracted by different methods

(图2.条带1:SDS法提取DNA;条带2:用SDS法提取得到DNA后,用Zymo纯化柱纯化DNA;条带3:常规方法提取DNA;M:DNAmarker)。(Fig. 2. Band 1: DNA was extracted by SDS method; Band 2: After DNA was extracted by SDS method, the DNA was purified by Zymo purification column; Band 3: DNA was extracted by conventional method; M: DNA marker).

Figure BDA0002557723370000101
Figure BDA0002557723370000101

表2不同方法提取获得的DNA的吸光值Table 2 Absorbance values of DNA extracted by different methods

(图3.条带1、2:表示用CTAB法提取得到的DNA;条带3、4:表示用SDS法提取得到的DNA;M:DNAmarker分子量同图2)(Figure 3. Bands 1 and 2: represent DNA extracted by CTAB method; Bands 3 and 4: represent DNA extracted by SDS method; M: The molecular weight of DNAmarker is the same as in Figure 2)

Figure BDA0002557723370000102
Figure BDA0002557723370000102

表3不同方法提取获得的DNA的吸光值Table 3 Absorbance values of DNA extracted by different methods

(图4.条带1:用滤膜法收集藻细胞;条带2:离心法收集藻细胞,用瓷珠打碎细胞;条带3、4:用过滤器收集藻细胞后用CTAB缓冲液裂解藻细胞;条带5、6:与条带3、4相同,添加了RNA酶;M:DNAMarker分子量同图2)。(Fig. 4. Band 1: algal cells were collected by filter method; band 2: algal cells were collected by centrifugation, and the cells were broken with ceramic beads; bands 3, 4: algal cells were collected by filter and then treated with CTAB buffer Lysate algal cells; Bands 5 and 6: the same as Bands 3 and 4, with the addition of RNase; M: the molecular weight of DNAMarker is the same as in Figure 2).

以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。The above-mentioned embodiments only represent several embodiments of the present invention, and the descriptions thereof are specific and detailed, but should not be construed as a limitation on the scope of the invention patent. It should be pointed out that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can also be made, which all belong to the protection scope of the present invention.

Claims (6)

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;
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 into a new centrifuge tube, adding RNase, and carrying out warm bath 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.
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: cADR G50, wherein CAIndicating 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.
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CN112322500A (en) * 2020-11-17 2021-02-05 江苏海洋大学 Aseptic processing method of Heteroflexus alba
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CN118147130B (en) * 2024-05-13 2024-08-02 中国科学院海洋研究所 A marine microalgae DNA extract and its application and rapid extraction method

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