KR20180107911A - A method of species identification and analysis of copepoda using a non-fracture type dna extraction - Google Patents

Abstract

The present invention relates to a method of species identification and form analysis of Copepoda using DNA extraction, which comprises the following: a preprocessing of preprocessing a biological sample; a hemolysis and clarifying step of adding a hemolytic buffer solution to the preprocessed biological sample; and a separation step of obtaining a cell hemolysis solution from the biological sample. An objective of the present invention to provide a method for simultaneously performing DNA extraction and morphological analysis in a single organism.

Description

Identification and Analysis Method of Copepods by Non - Fragmented DNA Extraction METHOD OF SPECIES IDENTIFICATION AND ANALYSIS OF COPEPODA USING A NON-FRACTURE TYPE DNA EXTRACTION}

The present invention relates to a species identification and analysis method capable of performing morphological analysis and gene analysis using only a single individual through the extraction of non-disruptive DNA of copepods.

Copepoda is an arthropod belonging to the arthropod crustaceans living in the sea, lake, river, etc. It is also called small crustacean. Copepods are underwater life, and representative species include gypsy plankton, Gyno copepods, and muddy water fleas. Recently, a method for evaluating environmental risk and the like by using copepods living in the offshore coast has been developed (Korean Patent No. 10-1088456).

On the other hand, it is common to observe copepods using an optical microscope for morphological studies of copepods. At this time, it is easy to observe the optical microscope. Therefore, a sample of copepods is immersed in lactic acid or lactophenol for a certain period of time, and the internal tissue is removed to make the specimen transparent.

However, through this process, the DNA of the copepod specimen is damaged and DNA can no longer be extracted from the specimen. Furthermore, since the size of the copepods is as small as 1 mm, it is difficult to extract DNA from only a part of the copepods.

For this reason, the morphological analysis and DNA analysis of copepods have been carried out by using specimen for morphological analysis and specimen for genetic analysis separately. In other words, more than 2 individuals were used for morphological analysis and DNA analysis of copepods.

However, in the case of sibling species or Cryptic species which are difficult to distinguish morphologically, morphological analysis and genetic analysis must be performed at the same time.

Therefore, in studying more accurate copepods and DNA analysis methods, the inventors of the present invention have found that DNA extraction from a single individual can be performed by separating a buffer portion for DNA extraction and a specimen for morphological observation after haemolysis and transparency without breaking the specimen And that the morphological analysis can be performed at the same time.

It is an object of the present invention to provide a method for simultaneously performing DNA extraction and morphological analysis in a single organism.

In order to achieve the above object,

Pretreating the biological specimen;

Hemolysis and transparency of a biological specimen by adding a cytolytic buffer solution to the pretreatment biological specimen; And

And separating the cell hemolysis solution from the biological sample.

DNA isolation and identification of species.

The present invention can perform morphological analysis and DNA extraction from a single copepod species. Thus, the present invention makes it possible to secure the base sequence of a holotype sample.

1 is a schematic diagram schematically showing the morphological analysis and DNA extraction method of the present invention.
FIG. 2 and FIG. 3 are dissecting microscope photographs of specimens before cell hemolytic buffer solution treatment.
FIG. 4 is an optical microscope photograph of a sample before treatment with cell hemolytic buffer solution.
Figs. 5 and 6 are a dissecting microscope photograph of a slide specimen of Example 1. Fig.
7 is an optical microscope photograph of a slide specimen of Example 1. Fig.

According to the present invention,

Pretreating the biological specimen;

Hemolysis and transparency of a biological specimen by adding a cytolytic buffer solution to the pretreatment biological specimen; And

And separating the cell hemolysis solution from the biological sample.

DNA extraction and identification of species.

Hereinafter, the present invention will be described in detail.

Biological specimen

The biological sample of the present invention belongs to the subphylum subclass. The biological specimen of the present invention may be any copepod, and is not particularly limited. That is, if the sample belongs to a copepod, the morphological analysis and DNA extraction can be performed through the method of the present invention, and the specific species of the sample is not limited in carrying out the present invention. For example, the copepods of the present invention can be copycat species living in coastal waters of Korea, such as Paracalanus parvus, Centropages abdominalis, Acartia omorii, Acartia erythraea, but are not limited thereto. Preferably, the biological sample of the present invention is a copepod with a size of not more than 2 mm, more preferably not more than 1 mm, but this is only an example of copepods for which the effect of the present invention is particularly useful. .

Preprocessing step

The morphological analysis and DNA extraction methods of the present invention comprise a pre-treatment step of a biological specimen. The pretreatment step may include washing the biological specimen. However, the pretreatment step does not include a process of damaging the external shape of the biological specimen. For example, the pretreatment step of the present invention does not include processes such as crushing, cutting, and the like of a biological specimen. Therefore, the biological sample of the present invention is used without being pulverized.

For example, the pretreatment step of the present invention may include a step of immobilizing the biological specimen with ethanol, firstly washing the specimen with purified water, and removing the foreign substance using conventional means such as a vortex mixer. In this case, the first washing with distilled water may be performed by immersing the biological specimen in distilled water for 30 minutes to 60 minutes.

Hemolysis and Transparency of Biological Specimens

The morphological analysis and DNA extraction method of the present invention includes hemolysis and transparency of a biological specimen by adding a cell hemolytic buffer solution to the pretreatment biological specimen. The cytolytic buffer solution is a solution for hemolyzing and clarifying a biological specimen. As a result, the internal structure of the biological specimen is melted and becomes transparent, which is advantageous for microscopic observation, and the DNA derived from the biological specimen is contained in the cell hemolytic buffer solution. That is, the outer shape of the biological specimen is not damaged and the inner tissue is melted by the hemolysis and transparency steps.

Preferably, the step of hemolyzing and clarifying the biological sample can be performed by adding a cell hemolyzing buffer solution to the moult sample and warming it at 54 to 58 ° C for 2 to 5 hours using an incubator.

The cell hemolysis buffer solution is not particularly limited as long as it is a solution that does not damage the external shape of the biological specimen but hemolyzes and transparentizes the biological specimen. For example, the cytolytic buffer solution may be a mixture of ATL buffer and proteinase. The protease is preferably Proteinase K. The mixture may be prepared by mixing protease: ATL buffer at a volume ratio of 1: 6 to 11, preferably 1: 9. ATL buffer disrupts cells to lyse cellular components, and Proteinase K degrades dissolved proteins in cells.

Separation step

The morphological analysis and DNA extraction method of the present invention includes a step of taking the cell hemolysis solution from the biological specimen. And the biological sample and the cell hemolysis solution are substantially separated by the separation step. Due to technical limitations, the cell hemolysis solution residue may remain in the biological sample, but this is not a significant level, and a sufficient amount of the hemolytic solution for DNA extraction is separated by the separation step. This allows the biological specimen to be used to observe the morphology of the biological specimen and extract the DNA from the cell hemolytic buffer solution taken. The separation step is performed with care to prevent damage to the biological specimen.

DNA recovery step

The morphological analysis and DNA extraction method of the present invention may further comprise a DNA recovery step of obtaining a DNA derived from a biological sample from the cell hemolytic buffer solution taken out after the separation step.

According to the hemolysis and transparency of the biological specimen, the hemolytic buffer solution contains DNA derived from biological specimens. In the DNA recovery step, the DNA is recovered by a conventional method. The conventional method may use centrifugation, but is not limited thereto.

Observation specimen preparation step

The morphological analysis and DNA extraction method of the present invention may further comprise an observational sample preparation step of preparing an observation specimen using the biological specimen after the separation step. The observation specimen is a specimen for observing the morphology of the biological specimen. For example, in the step of preparing the sample for observation, the biological specimen is separated from the cell hemolysis buffer solution, distilled water is added to the biological specimen, and the biological specimen is immersed in the distilled water for 30 to 60 minutes. Thereafter, the distilled water is removed and glycerol or lactophenol lactophenol) can be used to prepare a slide specimen. Then, after observing the entire portion using the observation specimen, the annex can be dissected to perform attachment observation.

For example, biological specimens are separated from cell hemolysis buffer solution, and then biological specimens are washed by adding distilled water to biological specimens. After dropping a few drops of lacto-phenol on the slide, place the specimen on it, position the specimen by the anatomical needle, and stack the cover slide on one side to match the height of the specimen. Carefully cover the new cover slide on the specimen. While moving the slides on the specimen, position the specimen in the desired direction and observe under the microscope.

After the complete sub-observation is completed, the cover slides are removed and the anatomical dissection is performed. The order of the dissection does not matter much, but it is normal, the tail -> 5 bastard -> 4 bastard -> 3 bastard -> 2 bastard -> 1 bastard -> 1 tactile -> 2 tactile -> gyrus (Daeak, ), And then dissect it in the following order. A few drops of lacto-phenol are dropped onto a new slide, and the attachment is transferred onto the lacto-phenol using an anatomical needle. Carefully cover the cover slides. When making a permanent sample, apply the cover slide edge with a transparent nail polish. Once the nail polish is firm, reapply it and let it dry. This operation is repeated two to three times to perform attachment observation.

At this time, when 100% glycerol is used directly, osmotic pressure may shrink the biological specimen, so that the concentration can be gradually increased from a low concentration to a high concentration.

Identification and morphological analysis method of the present invention by DNA extraction

The sibling identification and morphological analysis method of DNA extraction of the present invention is a DNA extraction and morphological analysis method of a non-crushed biosample without breaking the biosample. Therefore, the morphological analysis and DNA extraction method of the biological sample of the present invention can simultaneously perform DNA extraction and morphological analysis using a single biological entity. This is a technical feature of the present invention in which the biological sample of the DNA extraction sample and the biological sample of the morphological observation sample are distinguished from the conventional morphological analysis and DNA extraction method of the biological sample by crushing the biological sample and extracting the DNA from the crushed sample .

Thus, the present invention provides a method for obtaining a nucleotide sequence of a holotype sample (a perfect sample is a sample designated as a representative sample of the species by a researcher who has discovered a new species, and there is only one individual in the world) It is possible. In addition, the present invention is highly applicable when the number of samples is small and it is difficult to divide a sample for analysis of shape and a sample for analysis of gene. In addition, since the present invention can identify the DNA barcode system without analysis by nucleotide sequence analysis when it is constructed, the species can be identified through sequence analysis before the morphological analysis (DNA barcode technique is used to identify species Is a method for standardizing the identification of species using a very short base sequence of a specific region on the genome (a species having a low species variation and a large species variation). Therefore, the method of the present invention can be used to compare the base sequence on the DNA barcode system with the base sequence of the sample, and if the base sequences are different from each other, the morphology analysis of the sample (i.e., individual) .

BRIEF DESCRIPTION OF THE DRAWINGS The advantages and features of the present invention, and the manner of achieving them, will be apparent from and elucidated with reference to the embodiments described hereinafter in conjunction with the accompanying drawings. It should be understood, however, that the invention is not limited to the disclosed embodiments, but is capable of many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, To fully disclose the scope of the invention to those skilled in the art, and the invention is only defined by the scope of the claims.

≪ Materials and methods >

In the following test, females of the Centropages abdominalis species belonging to the long nodule Nannobidae were used. The C. abdominalis was harvested at the beach using a 200 um mesh net. After the collection, the sample was fixed with 95% ethanol so that the final concentration including the sample was 70% or more.

Cell lysis buffer was prepared by adding 20ul of Proteinase K to 180ul of ATL buffer. For DNA extraction, DNeasy Blood & Tissue Kit from QIAGEN was used. The basic protocol was based on the protocol of Qiagen's DNeasy Blood & Tissue Kit, which was partially modified.

<Example 1> Analysis of morphology and DNA extraction of non-fractured copepods

&Lt; Example 1-1 > A pretreatment step (i.e., washing step)

The primary distilled water was placed in a 1.5 ml tube, and a sample (C. abdominalis) fixed to ethanol was added thereto, and the tube was soaked for 50 minutes. Then, the foreign substances attached to the specimen were removed by using a Voltex mixer.

<Example 1-2> Hemolysis and transparency step

A new 1.5 ml tube was prepared, and cell lysis buffer was added thereto. Then, the specimen contained in the first distilled water was taken out from the first distilled water using a sterilized dissection needle and placed in a tube containing the hemolysis buffer solution. At this time, the sample was completely immersed in the cell hemolysis buffer solution.

The cell hemolytic buffer solution containing the sample was incubated at 56 ° C for 4 hours in an incubator. Transparent specimens were identified during the incubation period.

<Example 1-3> Separation of cell hemolytic buffer solution and sample

The cell hemolytic buffer solution was extracted from the cell hemolytic buffer solution tube under a stereomicroscope using a pipette and transferred into a new 1.5 ml tube. At this time, care was taken not to damage the specimen, and only cell hemolysis buffer solution was extracted.

<Example 1-4> Preparation of slide specimen

In the <Example 1-3>, the primary distilled water was placed in the tube where the specimen was left and immersed for 40 minutes. Then, the distilled water remaining in the tube was taken out, and a slide specimen was made using lacto phenol. Then, a slide specimen was observed using a microscope.

Example 1-5 DNA recovery

200 μl of AL buffer was added to the cell hemolysis buffer solution contained in the new tube obtained in the above <Example 1-3> and mixed well. Then, 200 ul of 99 v / v% ethanol was added and mixed well. Then, the cell hemolysis buffer solution to which the AL buffer and ethanol were added was put into a 2 ml column tube and centrifuged at 8,000 rpm for 1 minute. The separated solution was discarded and 500 ul of AW 1 buffer was added to the column tube and centrifuged at 8,000 rpm for 1 minute. The separated solution was discarded again and 500 μl of AW 2 buffer was added to the column tube and centrifuged at 14,000 rpm for 3 minutes. The separated solution was discarded again. 100 μl of tertiary distilled water was added to the column tube, and the DNA was recovered by centrifugation at 14,000 rpm for 1 minute.

Here, Examples <1-1> to <1-5> were performed three times using three samples (C. abdominalis).

&Lt; Control group >

As a control, DNA was extracted from C. abdominalis using a Qiagen kit. This is a conventional copepod DNA extraction method. The specific DNA extraction method is as follows.

A sample (C. abdominalis) fixed to ethanol was taken out and shredded using a grinder. The crushed specimen was placed in a 1.5 ml tube containing the cell hemolytic buffer solution. Then, the cell hemolytic buffer solution tube containing the above-mentioned main sample was put into an incubator and incubated at 56 ° C for 4 hours.

After incubation, 200 ul of AL buffer was added to the tube and mixed well. Then, 200 ul of 99 v / v% ethanol was added to the tube containing the crushed specimen, cell hemolytic buffer solution and AL buffer, and mixed well.

Then, the cell hemolysis buffer solution to which the AL buffer and ethanol were added was put into a 2 ml column tube and centrifuged at 8,000 rpm for 1 minute. The separated solution was discarded and 500 ul of AW 1 buffer was added to the column tube and centrifuged at 8,000 rpm for 1 minute. The separated solution was discarded again and 500 μl of AW 2 buffer was added to the column tube and centrifuged at 14,000 rpm for 3 minutes. The separated solution was discarded again. 100 μl of tertiary distilled water was added to the column tube, and the DNA was recovered by centrifugation at 14,000 rpm for 1 minute.

At this time, the control group was also performed three times using three samples (C. abdominalis).

&Lt; Experimental Example 1 >

The slide specimen obtained in Example 1 was photographed using a dissecting microscope (microscope) and an optical microscope, and the shape thereof was observed.

At this time, after the steps of the cell hemolysis buffer solution treatment, that is, the steps of Example 1-1, were completed, the specimens in which the foreign substances have been removed are also examined by a microscope.

As a result, in the samples before the treatment with the hemolytic buffer solution, it was difficult to observe the internal tissues accurately because the specimen was not transparent. Particularly, since the light microscope has light from below, the internal structure of the specimen is not transparent and light can not be transmitted, making it more difficult to observe accurately (Figs. 2 to 4, 2 and 3: dissecting microscope photograph, Figure 4: Optical microscope photograph).

On the other hand, in the case of the slide sample obtained in Example 1, since the samples were transparent through the treatment with the cell hemolytic buffer solution, observation was easy. In addition, the shape of the specimen was not damaged and the internal tissues were melted to be transparent (Figs. 5 to 7, Figs. 1 and 6: Anatomical microscope photograph, and Fig. 7: Optical microscope photograph).

<Practical Example 2> DNA extraction efficiency evaluation

The concentration of DNA extracted from Example 1 and the control group was measured. As a result, it was confirmed that there was no significant difference in DNA concentration between Example 1 and the control group (Table 1).

Example 1 Control group DNA concentration
(ng / ul) 10.19 13.63 16 11.82 14.41 10.77

Claims (10)

Pretreating the biological specimen;
Hemolysis and transparency of a biological specimen by adding a cytolytic buffer solution to the pretreatment biological specimen; And
And separating the cell hemolysis solution from the biological sample.
Identification of species by DNA extraction and morphometric analysis.
The method according to claim 1,
Wherein said organism is a copepod.
The method according to claim 1,
Wherein said cytolytic buffer solution hemolyzes and clarifies the biological specimen.
The method according to claim 1,
Further comprising a DNA recovery step of obtaining a DNA derived from a biological sample from the cell hemolysis buffer solution after the separation step.
The method according to claim 1,
Further comprising the step of preparing a sample for observation using said biological sample after said separation step.
The method according to claim 1,
After the separation step, the biological sample is observed using a biological specimen, and DNA is extracted from the obtained cell hemolytic buffer solution.
The method according to claim 1,
Wherein the biological sample is used without being pulverized.
The method according to claim 1,
Wherein the outer shape of the biological specimen is not damaged and the inner tissue is melted by the hemolysis and transparency step.
The method according to claim 1,
Characterized in that the method is carried out using a single biological sample
The method according to claim 1,
Wherein the step of pretreatment of the biological specimen includes a step of washing the biological specimen and the step of impairing the external form of the biological specimen is not included

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