CN117126843A - DNA extraction method for small zooplankton single body - Google Patents
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Abstract
The invention discloses a DNA extraction method for a small zooplankton single body, which relates to the technical field of zooplankton DNA extraction, and comprises the following steps in sequence: (1) Fixing the collected small zooplankton with neutral Lugol's solution, transferring to a counting frame, flushing, sucking the small zooplankton single body, placing in a PCR tube, and airing; (2) Adding DNA lysate for cracking, hot compressing, embedding PCR tube into a heat preservation box, placing under an ultrasonic homogenizer, and performing ultrasonic crushing; (3) Heating in water bath immediately, then placing under an ultrasonic homogenizer, and performing ultrasonic crushing again; (4) And hot compress the PCR tube after ultrasonic crushing again, putting the PCR tube on ice, rapidly freezing, taking out the PCR tube to room temperature, and centrifuging the PCR tube to obtain the DNA of the small zooplankton single body. The invention can greatly reduce the DNA loss in the extraction process, and effectively solves the problems of difficult shell and cell disruption, low DNA concentration purity and the like in the extraction process in the prior art.
Description
Technical Field
The invention relates to the technical field of zooplankton DNA extraction, in particular to a DNA extraction method for a small zooplankton single body.
Background
Phylogenetic geography is a subject for researching historical reasons and evolution processes of the existing distribution patterns formed by different species in species, and the formation mechanism, the phylogenetic relation and the existing distribution characteristics of the intra-species system geography are discussed through analysis of the genetic structure of the species, so that the evolutionary history of the species is traced and revealed, and the phylogenetic geography has important significance for understanding the processes of species differentiation, species formation, ecological adaptation, historical climate change and the like. As the bodies of small zooplankton such as rotifers are about 50-2000 mu m long, the zooplankton has the characteristics of global distribution, relatively short generation time, response to environmental changes and rapid occupation of ecological niches, and has been widely used as model organisms for biophysical researches. Species genomic DNA acquisition is an important link in developing the study, however rapid and efficient extraction of DNA from individual small zooplankton is one of the major problems restricting the rapid development of the study. Because of low individual small DNA concentration of small animals, the traditional small zooplankton DNA extraction method adopts field collection, single bodies are separated in a laboratory for cloning culture, and when the density of each cloning body reaches about 200, DNA extraction and subsequent PCR are carried out. However, many species are difficult to perform in laboratory culture, and thus small zooplankton DNA extraction of individual bodies is necessary.
At present, three methods for single individual DNA of zooplankton at home and abroad are mainly available: one is the Chelex-100 chelate resin method proposed by Walsh et al, which was originally used to extract DNA from forensic materials. The extraction process of the method is to add a lysate, then to add Chelex-100 emulsion resin particles (InstaGeneMatrix; bio-Rad, hercules, calif., U.S. A) produced by Bio-Rad, inc. of America, to break up organisms, followed by a thermal incubation to obtain genomic DNA. The improved kit for extracting genome DNA of micro-tissues and individuals based on the method is Qiagen DNeasy tissue extraction kit manufactured by Qiagen, germany, genomic DNA purification kit manufactured by Promega, U.S.A. The basic principle of commercial kits is that the collection and purification of DNA are completed through adsorption and elution of membranes, and besides the expensive reagent, the method mainly has the following problems: 1. because the small zooplankton individuals are small, the total content of DNA is far less than that of the traditional DNA extraction sample, and DNA is further lost through centrifugal column membrane or resin adsorption and elution, so that the downstream PCR experiment is affected. 2. Resin particles contained in the Chelex-100 reagent, which result in the extraction of total DNA impurities, have been shown in practice to inhibit the downstream PCR reactions to varying degrees. The second method is the HotSHOT method proposed by Montero-Pau et al, which was originally used for zooplankton resting egg DNA extraction. The method is an alkaline lysis method, wherein alkaline lysate is added, then the alkaline lysate is heated at a high temperature of 95 ℃ for half an hour, and then the alkaline lysate is placed on ice for cold shock to release DNA, and finally acidic buffer solution is added to adjust the pH to be neutral. This method is cheaper and simpler than the chelate resin method, but has a disadvantage that a stable DNA extract cannot be produced. However, in the method, alkaline lysate is added to release DNA, and then an equal volume of acidic buffer solution is used to adjust the pH to be neutral, so that the final pH is easy to be unstable, and the long-term preservation of the DNA is not facilitated. In addition, disruption of biological structures using tools such as a gun head and a dissecting needle tends to leave biological tissues on the tools, resulting in a huge loss of DNA in the individual samples having low DNA content per se. The third method is Wu Lisheng, etc. to clean and purify DNA precipitate, and is mainly used for extracting single copepod resting eggs. The method comprises the steps of adding proteinase K and buffer solution for crude extraction, adding a nucleic acid coprecipitation agent for separation, discarding supernatant and cleaning. Unlike resting eggs, small zooplankton such as Desmodium and the like have shells, so that DNA is difficult to dissolve or break down by adding only lysate, proteinase K and the like to release, and the external physical means are necessary to destroy the shells of individuals.
The key point of DNA extraction of the single body of the small zooplankton is as follows: 1. effectively destroying shells and cells of small zooplankton and releasing DNA; 2. the DNA content of the individual body is low, and the DNA loss caused by the operation processes of tube replacement, cleaning and the like is reduced as much as possible. Therefore, a small zooplankton single-body DNA extraction method which can effectively damage the shell of the small zooplankton, has less damage to the integrity of DNA, has less loss of DNA content, is convenient and cheap and solves the technical difficulties, so as to meet the research of phylogenetic geography.
Disclosure of Invention
In order to solve the technical problems, the invention aims to provide a DNA extraction method for a small zooplankton single body, which can greatly reduce the DNA loss in the extraction process and effectively solve the problems of difficult shell and cell disruption, low DNA concentration purity and the like in the extraction process in the prior art.
The technical scheme for solving the technical problems is as follows: the DNA extraction method for the small zooplankton single body comprises the following steps in sequence:
(1) Fixing the collected small zooplankton with neutral Lugol's solution, separating and transferring the individual to a sterile 1mL counting frame by using a capillary glass tube under a stereoscopic microscope, flushing for 2-3 times by using sterile ultrapure water, sucking the small zooplankton individual by using the capillary glass tube, placing the small zooplankton individual into a PCR tube, and finally adding absolute ethyl alcohol for airing;
(2) Adding DNA lysate into PCR tube for cracking, hot compressing at 50-60deg.C for 4-6 hr for softening, embedding PCR tube into heat preservation box filled with ice, placing under ultrasonic homogenizer, embedding ultrasonic probe beside the wrapped PCR tube, and ultrasonic crushing;
(3) Immediately placing the ultrasonic-crushed PCR tube in a water bath at 80 ℃ for heating, then placing under an ultrasonic homogenizer, burying an ultrasonic probe beside the PCR tube, and performing ultrasonic crushing again;
(4) And hot compress the crushed PCR tube at 95 ℃ for 30min, then rapidly freezing the PCR tube on ice at-80 ℃ for 10min, taking out, and centrifuging at room temperature to obtain the small zooplankton single body DNA.
Further, in the step (1), the final concentration of the neutral Lugol solution is 1-3wt%.
Further, in step (2), the DNA lysate was Tris-HCl 9-11mM, na 2 EDTA 0.4-0.6mM, the remainder ddH 2 O, pH 8.0.
Further, in step (2), the DNA lysate was Tris-HCl 10mM, na 2 EDTA 0.5mM, the remainder ddH 2 O, pH 8.0.
Further, in the steps (2) and (3), the ultrasonic crushing is carried out for 5 minutes under the conditions of 20-40kHz and 25-40W.
The invention has the following beneficial effects:
1. the extraction method can greatly reduce the DNA loss in the extraction process, and effectively solves the problems of difficult shell and cell disruption, low DNA concentration purity and the like in the extraction process in the prior art.
2. The invention uses ultrasonic breaking technique to break small zooplankton, to break small zooplankton shell and cell and release DNA. On one hand, the ultrasonic waves effectively crush plankton such as dinoflagellate, rotifer and the like with thick walls or shells; on the other hand, the method can reduce the chance of external pollution caused by tool operation, DNA adsorbate addition and the like, and greatly reduce DNA loss caused by residual organism tissues on the tool, centrifugal column filtration and the like.
3. The invention uses ultrasound at low temperature, and the main purpose is to strengthen zooplankton shells and tissue brittleness through low temperature, so that the zooplankton shells and tissue brittleness are fully crushed under the ultrasound. The main purpose of the ultrasound at high temperature is to make the cytoplasm more easily flow out at high temperature, and the protein is denatured so that the DNA is fully released.
4. The DNA lysate adopted by the invention only contains Tris-HCl and Na 2 EDTA, SDS, CTAB, proteinase K and other matters are not contained, so that the DNA extracting solution can be used as template for downstream PCR directly without washing, filtering and other operations. And the DNA lysate with pH value of 8 is adopted to replace the method of adding alkaline lysate and then adding acid buffer solution to adjust the pH value, so that the DNA buffer environment is more stable, and the long-term preservation of the DNA is facilitated.
5. The invention is completed in a PCR instrument and an ultra-low temperature refrigerator respectively by hot compress at 95 ℃ and cold shock at low temperature, and is simpler and more convenient than the traditional method for extracting DNA by adopting water bath hot compress and liquid nitrogen cold shock.
Drawings
FIG. 1 shows the result of gel electrophoresis of the PCR products in example 1;
FIG. 2 shows the result of gel electrophoresis of the PCR products in example 2;
FIG. 3 is a diagram of an electrophoresis gel in comparative example 1.
Detailed Description
The principles and features of the present invention are described below with examples given for the purpose of illustration only and are not intended to limit the scope of the invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Example 1
Extracting single individual genome DNA of 15 small zooplankton by using the method, using the extracted single individual genome DNA as a template, directly using the single individual genome DNA for PCR experiments, and successfully amplifying mitochondrial DNA cytochrome oxidase c oxidase subunit I fragment (COI) genes by using universal primers; the specific process is as follows:
a DNA extraction method for small zooplankton single bodies sequentially comprises the following steps:
(1) Collecting zooplankton samples in a wetland in Guangzhou city by using a 64 μm aperture plankton trawl, fixing the collected small zooplankton with neutral Rugosa liquid with the final concentration of 2wt%, separating and transferring a single individual to a sterile 1mL counting frame by using a capillary glass tube under a stereoscopic microscope, flushing sterile ultrapure water for 3 times until no other organisms or pollutants are attached to the body surface, selecting a single individual of a sepia armigera, a large-belly foot wheel worm, a spiral tortoise shell wheel worm, a copepod nauplii, a gyrotria, a wheel worm, a long limb multi-limb wheel worm and a wide-spread multi-limb wheel worm by using the capillary glass tube, placing the single individual into the PCR tube, and finally adding 5 mu L of absolute ethyl alcohol for airing to remove redundant water;
(2) Adding 25 μL of DNA lysate into each PCR tube, performing pyrolysis, hot compressing at 55deg.C for 5 hr for softening, embedding the PCR tubes into ice-filled thermal insulation box, and placing in ultrasoundUnder the wave homogenizer, an ultrasonic probe is buried beside the wrapped PCR tube, and ultrasonic crushing is carried out for 5min under the conditions of 20kHz and 25W; the DNA lysate was Tris-HCl 10mM, na 2 EDTA 0.5mM, the remainder ddH 2 O, pH 8.0;
(3) Immediately placing the ultrasonic-crushed PCR tube in a water bath at 80 ℃, then placing under an ultrasonic homogenizer, burying an ultrasonic probe beside the PCR tube, and ultrasonic-crushing for 5min again under the conditions of 20kHz and 25W;
(4) And hot compress the crushed PCR tube at 95 ℃ for 30min, then rapidly freezing the PCR tube on ice at-80 ℃ for 10min, taking out, and centrifuging at room temperature to obtain the small zooplankton single body DNA.
A PCR reaction system was prepared by taking 5. Mu.L of the above-obtained small zooplankton single-body DNA as a template according to the TaKaRaex Taq protocol. The COI fragment (660 bp) was amplified using the COI universal primer LCOI 1490 (5'-GGTCAACAAATCATAAAGATATTGG-3'), HCOI 2198 (5'-TAAACTTCAGGGTGACCAAAAAATCA-3') (Folmer et al, 1994).
Setting a PCR process: pre-denaturation at 94℃for 3min, followed by 35 cycles (denaturation at 94℃for 30s, annealing at 52℃for 30s, extension at 72℃for 45 s) and extension at 72℃for 8min after the end of the cycle. The PCR products were detected by gel electrophoresis as shown in FIG. 1. Wherein, 1-2 is the PCR result of the single genome DNA of the Torulopsis megalobus, 3 is the PCR result of the single genome DNA of the Torulopsis megalobus, 4-5 is the PCR result of the single genome DNA of the Torulopsis megalobus, 6-7 is the PCR result of the single genome DNA of the Torulopsis megalobus, 8-9 is the PCR result of the single genome DNA of the Torulopsis megalobus, 10-11 is the PCR result of the single genome DNA of the Torulopsis, 12-13 is the PCR result of the single genome DNA of the Torulopsis megalobus, 14-15 is the PCR result of the single genome DNA of the Torulopsis megalobus, and 16 is the negative control.
As can be seen from FIG. 1, 15 individuals subjected to the experiment all obtain target bands, which indicates that the individual genomic DNA obtained by the extraction method of the invention can be directly applied to downstream PCR reaction and is effective for different small zooplankton.
Example 2
The invention is used for extracting genome DNA of the small zooplankton widely-distributed multi-limb rotifer, the obtained genome DNA is used as a template, and the nuclear genome 18SrRNA gene and the 28S rRNA gene are successfully amplified by using universal primers; the specific process is as follows:
a DNA extraction method for small zooplankton single bodies sequentially comprises the following steps:
(1) Collecting zooplankton samples in a wetland in a city of Guangzhou by using a 64 μm aperture plankton trawl, fixing the collected small zooplankton with neutral Rug's solution with the final concentration of 2wt%, separating and transferring the wide-spread multi-limb artemia into a sterile 1mL counting frame by using a capillary glass tube under a stereoscopic microscope, flushing the sterile ultrapure water for 3 times until no other organisms or pollutants are attached to the body surface, sucking a single wide-spread multi-limb artemia by using the capillary glass tube, placing the single wide-spread multi-limb artemia into a PCR tube, and finally adding 5 mu L of absolute ethyl alcohol for airing to remove excessive moisture;
(2) Adding 25 mu L of DNA lysate into each PCR tube for pyrolysis, hot compressing for 5 hours at 55 ℃ for softening, then embedding the PCR tubes into a heat preservation box filled with ice and placing under an ultrasonic homogenizer, embedding an ultrasonic probe beside the wrapped PCR tubes, and carrying out ultrasonic crushing for 5 minutes under the conditions of 20kHz and 25W; the DNA lysate was Tris-HCl 10mM, na 2 EDTA 0.5mM, the remainder ddH 2 O, pH 8.0;
(3) Immediately placing the ultrasonic-crushed PCR tube in a water bath at 80 ℃, then placing under an ultrasonic homogenizer, burying an ultrasonic probe beside the PCR tube, and ultrasonic-crushing for 5min again under the conditions of 20kHz and 25W;
(4) And hot compress the crushed PCR tube at 95 ℃ for 30min, then rapidly freezing the PCR tube on ice at-80 ℃ for 10min, taking out, and centrifuging at room temperature to obtain the small zooplankton single body DNA.
According to TaKaRa ex Taq scheme, 4 PCR tubes were used to prepare PCR reaction system. Wherein, no. 1-3 takes 5. Mu.L of the extracted individual DNA as a template, and No. 4 takes 5. Mu.L of sterile ultra-pure water as a negative control. 1-3 were amplified using the ribosomal RNA encoding gene 18S rDNA V4 region universal primer (320 bp), 28S rDNA D2 region universal primer (360 bp) and 18S rDNA V4-V5 region universal primer (490 bp), respectively:
528F(5’-GCGGTAATTCCAGCTCCAA-3’)，
706R(5’-AATCCRAGAATTTCACCTCT-3’)；
LSU335(5’-ACCGATAGCRAACAAGTA-3’)，
LSU714(5’-TCCTTGGTCCGTGTTTCA-3’)；
18SCOMF(5’-TGCATGGCCGTTCTTAGTTGGTGG-3’)，
18SCOMR(5’-CACCTACGGAAACCTTGTTACGAC-3’)；
PCR reaction conditions: pre-denaturation at 94℃for 3min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 30s, and cycle number of 35; finally, the extension is carried out for 10min at 72 ℃.
The PCR products were detected by gel electrophoresis as shown in FIG. 2.
As can be seen from FIG. 2, the above 3 general primers were successful in PCR amplification of the single genomic DNA extracted by the method and obtaining the target band, indicating that the small zooplankton single genomic DNA extracted by the method of this example can be used as a template for both mitochondrial genome fragment amplification and nuclear genome fragment amplification.
Comparative example 1
Based on the experimental procedure of example 1, comparing the results of PCR amplification by the COI universal primer of DNA products extracted by four methods, namely, using a stereoscopic vision under-dissecting needle invasive type crushing, low-temperature ultrasound+high-temperature ultrasound crushing, high-temperature ultrasound crushing only, and low-temperature ultrasound crushing; the electrophoresis gel diagram is shown in FIG. 3.
As can be seen from fig. 3, the target band can be obtained by both the dissecting needle invasive disruption (1) and the low temperature+high temperature ultrasonic disruption (2), but the invasive disruption involves a risk of introducing foreign DNA contamination due to human operation differences. The use of high temperature sonication alone (3) may fail to disrupt the harder parts of the individual's housing, etc., failing to allow cell efflux and adequate release of DNA, resulting in the absence of target bands. The target bands were only visualized by low temperature sonication (4), but the brightness was very low, which was related to the lower concentration of DNA template. Experiments prove that the method for extracting the single individual genome DNA by adopting the low-temperature and high-temperature ultrasonic disruption method can replace a foreign object tool invasive disruption mode, and the obtained DNA concentration can be directly applied to a downstream PCR experiment.
The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.
Claims (5)
1. A DNA extraction method for a small zooplankton individual, comprising the steps of, in order:
(1) Fixing the collected small zooplankton with neutral Lugol's solution, separating and transferring the individual to a sterile 1mL counting frame by using a capillary glass tube under a stereoscopic microscope, flushing for 2-3 times by using sterile ultrapure water, sucking the small zooplankton individual by using the capillary glass tube, placing the small zooplankton individual into a PCR tube, and finally adding absolute ethyl alcohol for airing;
(2) Adding DNA lysate into PCR tube for cracking, hot compressing at 50-60deg.C for 4-6 hr for softening, embedding PCR tube into heat preservation box filled with ice, placing under ultrasonic homogenizer, embedding ultrasonic probe beside the wrapped PCR tube, and ultrasonic crushing;
(3) Immediately placing the ultrasonic-crushed PCR tube in a water bath at 80 ℃ for heating, then placing under an ultrasonic homogenizer, burying an ultrasonic probe beside the PCR tube, and performing ultrasonic crushing again;
(4) And hot compress the crushed PCR tube at 95 ℃ for 30min, then rapidly freezing the PCR tube on ice at-80 ℃ for 10min, taking out, and centrifuging at room temperature to obtain the small zooplankton single body DNA.
2. A DNA extraction method for small zooplankton individuals as in claim 1 wherein in step (1) the final concentration of neutral lugwort is 1-3% by weight.
3. A DNA extraction method for small zooplankton single bodies according to claim 1, whereinIn the step (2), the DNA lysate is Tris-HCl 9-11mM, na 2 EDTA 0.4-0.6mM, the remainder ddH 2 O, pH 8.0.
4. A DNA extraction method for small zooplankton individuals as claimed in claim 1 or 3 wherein in step (2) the DNA lysate is Tris-HCl 10mm, na 2 EDTA 0.5mM, the remainder ddH 2 O, pH 8.0.
5. The method for DNA extraction of small zooplankton single bodies according to claim 1, wherein in the steps (2) and (3), the ultrasonic disruption is performed at 20 to 40kHz and 25 to 40W for 5 minutes.
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