KR101497525B1 - Method of extracting dna from marine organism - Google Patents

Abstract

The present invention relates to a novel method for rapidly and easily extracting pure DNA, which can be used for PCR analysis, on a trace amount of tissues and cells collected from marine aquatic organisms having vast genetic diversity. According to the DNA extraction method, the DNA extracted from a marine aquatic organism is cultivated in a dissolution buffer for DNA extraction containing a nonionic surfactant and a bead, and a step of inactivating the protease, It is able to provide high quality pure DNA that can be used for PCR quickly and easily within a maximum of 7 minutes from various fish marine animals and plants such as fish tissue samples, plant and animal plankton, and molluscan tissue samples. In addition to fresh tissues, The present invention can be applied to Since the PCR result of the same level as that immediately after the treatment is obtained even after storing the extracted DNA solution for a long period of time, it is possible to stably identify the gene repeatedly and thus it is expected to contribute to securing various gene information of marine aquatic organisms.

Description

METHOD OF EXTRACTING DNA FROM MARINE ORGANISM BACKGROUND OF THE INVENTION [0001]

The present invention relates to a method for extracting DNA from marine aquatic organisms, and more particularly, to a method for extracting DNA of marine aquatic organisms from a marine aquatic organism having a large genetic diversity, To a novel DNA extraction method using a tissue-dissolving buffer solution which can be rapidly and easily extracted.

Marine aquatic organisms are very important in terms of being an important protein supply source of mankind and at the same time possessing vast genetic diversity so that future useful gene resources can be discovered. In this regard, large-scale exploration of marine and marine bioscience materials is needed to prevent the extinction of marine aquatic organisms and to discover and use special function proteins and genes that can not be found on land.

Polymerase chain reaction (PCR) is the most commonly used molecular biologic method for these studies, which has the advantage that not only purely isolated DNA but also partially extracted DNA can be used. Therefore, there is a need for a technique for rapidly and easily extracting DNA from a large amount of samples without damaging the DNA, and in particular, in the case of high-priced and rare species, a technology capable of extracting DNA using only minimal tissue sample It is essential.

As a method of extracting DNA from marine aquatic organisms, a DNA extraction method including a process using phenol and chloroform, which are conventionally used in humans or mammals, is generally used. Microalgae are used for DNA extraction . These traditional methods require long periods of time and require concentration due to many steps, and the DNA recovery rate also depends greatly on the operator's skill, which can lead to erroneous results due to the manual labor of an inexperienced operator.

The conventional method of extracting DNA using phenol / chloroform or phenol / chloroform / isoamyl alcohol is a method of extracting DNA (or isopropanol) It takes a step and time, and there is a problem that phenol and chloroform which are harmful to the human body are used. In addition, the conventional plant DNA extraction method is a method in which a plant cell is treated with a chemical treatment method using an enzyme or a complex reagent (Holm, C., DW Meeks-Wagner, WL Fangman and D. Bostein, . yeast Gene 42;. 169-173; . Lippke, JA, Strzempko, MN, Raia, FF, Simon, SL and French, CK 1987. Isolation of intact high-molecular weight DNA by using guanidine isothiocyanate Applied and Environmental Microbiology . 53; 2588-2589; Verma, A. and Kwon-Chung, KJ 1991. Rapid Method to Extract DNA from Cryptococcus neoformans . Jounal of Clinical Microbiology . 29 (4); 810-812) or a physical treatment method using bead beating (Cardinali, G., Liti, G. and Martini, A. 2000. Non-radioactive dot-blot DNA reassociation for unequivocal yeast identification. International journal of systematic and evolutionary microbiology . 50; 931-936), and then DNA is separated using a membrane to which DNA is attached. However, this method has many drawbacks such as the time required for complete liquefaction of the microalgae takes about 10 minutes to 1 hour, and the subsequent extraction process is complicated and takes more than one hour and a half of time with many reagents.

Therefore, reproducible results can be obtained when repetitive experiments involving the treatment of a large amount of samples are required, and a simple and widely applicable method for extracting DNA from marine aquatic organisms having various sieve components (Wang, W., Wang, W., Wang, W., Wang, W. and Wang, -based DNA extraction from paraffin-embedded tissue for PCR amplification. Biotechnology . 18; 768; Estoup, A., Largiader, CR, Perrot, E. and Chourrout, D. 1996. Rapid one-tube DNA extraction for reliable PCR detection fish polymorphic markers and transgenes. Molecular Marine Biology and Biotechnology . 5: 295-298; Nelson, RJ, Beacham, TD and Small, MP 1998. Microsatellite analysis of the Vancouver Island sockeye salmon ( Oncorhynchus nerka ) stock complex using nondenaturing gel electrophoresis. Molecular Marine Biology and Biotechnology . 7: 312-319; Taris, N., Baron, S., Sharbel, TF, Sauvage, C. and Boudry, P. 2005. A combined microsatellite multiplexing and boiling DNA extraction method for high throughput parentage analyses in the Pacific Oyster ( Crassostrea gigas ). Aquaculture Research . 36; 516; Aranishi, F. and Okimoto, T. 2006. A simple and reliable method for DNA extraction from bivalve mantle. Journal of Applied Genetics . 47; 251-254).

In recent years, DNA extraction studies have been conducted on unique tissue samples of marine aquatic organisms. In particular, a method of extracting DNA from fins, blood, scales, oral epithelial cells, sperm, and eggs has been devised is (Cummings, and AS Thorgaard, GH 1994. Extraction of DNA from fish blood and sperm Biotechniques 17;.. 426; Adcock, GL, Ram, B. Hauser, Smith, LP and Carvalho, GR 2000. Screening of DNA polymorphisms in samples of archived scales from New Zealand snapper. Journal of Fish Biology . 56: 1283-1287; Sire, JY, Girondot, M. and Babiar, O. 2000. Marking zebrafish, Danio rerio ( Cyprinidae ), using scale regeneration. Journal of Experimental Zoology . 286: 297-304; Yue, GH and Orban, L. 2001. Rapid Isolation of DNA from Fresh and Preserved Fish Scales for Polymerase Chain Reaction. Marine Biotechnology . 3: 199-204; Nam, YK, Park, JE, Kim, KK and Kim, DS 2003. A rapid and simple PCR-based method for analysis of transgenic fish using a restricted amount of fin tissue. Transgenic Research . 12: 523-525; Wasko, AP, Martins, C., Oliveira, C. and Foresti, F. 2003. Nondestructive genetic sampling in fish. An improved method for DNA extraction from fish fins and scales. Hereditas . 138: 161-165; Aranishi, F. 2006. Single fish egg DNA extraction for PCR amplification. Conservation Genetics . 7: 153-156; Aranishi, F., Okimoto, T. and Ohkubo, M. 2006. A short-cut DNA extraction from cod caviar. Journal of the science of Food and Agrculture . 86: 425-428; Livia, L., Antonella, P., Hovirag, L., Mauro, N. and Panara, F. 2006. A nondestructive, rapid, reliable and inexpensive method to store and extract high-quality DNA from fish body mucus and buccal cells. Molecular Ecology Notes . 6: 257-260; Lopera-Barrero, NM, RP Ribeiro, RN Sirol, JA Povh, PC Gomes, L. Vargas and DP Streit Jr. 2006. Genetic diversity in piracanjuba populations ( Brycon orbignyanus ) with the RAPD (Random Amplified Polymorphic DNA) markers. Journal Animal Science . 84; 170; Kumar, R., Singh, PJ, Nagpure, NS, Kushwaha, B., Srivastava, SK and Lakra, WS 2007. A non-invasive technique for rapid extraction of DNA from fish scales. Indian jounal of experimental biology. 45: 992-997).

However, such a DNA extraction method has a disadvantage in that it can be used only for a tissue sample of a specific species, and the reliability of the result is low due to low reproducibility despite the use of an expensive reagent. In addition, some of these methods involve a complicated process, which not only takes a long time, but also affects the purity of the DNA due to contamination.

As a result of intensive efforts to solve the disadvantages of the prior art described above, the inventors of the present invention have devised a new buffer solution for tissue dissolution for use in DNA extraction, and have widely applied a variety of marine biological samples using this buffer As a result, it was confirmed that DNA can be extracted with high reproducibility through a short step in a short time, thereby completing the present invention.

It is an object of the present invention to provide a method for rapidly and easily extracting pure DNA using only a very small amount of tissues and cells from marine aquatic organisms having genetic diversity so as to efficiently perform PCR analysis on them .

Another object of the present invention is to provide a buffer composition for tissue dissolution which can be used for a method for rapidly and easily extracting DNA usable for PCR analysis from marine aquatic organisms.

In order to achieve the above object, according to the present invention,

The tissue or cells collected from marine aquatic organisms are cultured in a tissue-dissolving buffer containing non-ionic surfactants, beads and proteolytic enzymes,

And inactivating the proteolytic enzyme at 90 to 100 占 폚.

As the nonionic surfactant, NP40 (Nonidet P-40; Octylphenoxy polyethoxy ethanol) and Tween 20 are preferable, and they can be used in a ratio of 1: 1 in a concentration of 0.5 to 9.0% (v / v). The beads are preferably glass beads having a diameter of 1.0 to 50.0 탆 and can be used in an amount of 0.5 to 1.0% (v / v) in the buffer. It is also preferable to use protease K at a concentration of 50 to 150 占 퐂 / ml in the buffer solution.

In order to achieve the other object of the present invention, there is provided a buffer composition for DNA extraction of marine aquatic organisms, which comprises a non-ionic surfactant NP40 (Nonidet P-40) and Tween 20 in a ratio of 1: 1, Proteinase is an essential component and may further contain Tris-Cl, KCl and MgCl ₂ . To maintain the acidity of the buffer, Tris-Cl is 5 to 90 mM in concentration and contains 25 to 450 mM of KCl and 0.75 to 135 mM of MgCl ₂ to form a buffer suitable for rapid and efficient PCR analysis desirable.

A preferred buffer composition according to the present invention comprises 10 mM Tris-Cl; and a pH 8.0, 50 mM KCl, 1.5 mM MgCl 2, 1.0% NP 40, 1.0% Tween 20, the bead and protease K.

Hereinafter, the present invention will be described in detail.

The beads which can be used in addition to the buffer for DNA extraction according to the present invention refer to ordinary beads used for bead beating. The material of the beads is not particularly limited, but glass beads are preferably used. In the buffer solution of the present invention, the bead is used to reduce the time required for the sample to dissolve effectively. The amount of beads contained in the buffer solution is preferably 5 to 10 μg, and most preferably 0.5%. The size of the beads is not particularly limited, but it is preferable that the beads have a diameter of 1.0 to 50 탆, and most preferably beads having a diameter of 30 탆 are mixed and used.

Herein, the term bead beating refers to a method of physically breaking tissues and cell walls by rotating a bead using a bead and a vortex machine, and then applying a shock of the rotated bead. The rotation speed of the beads in the bead beating is not particularly limited, but can be performed at 1,400 to 3,200 rpm for 5 to 10 seconds. The bead beating of the present invention is intended to facilitate the detergent to rapidly dissolve the cell membrane by slightly damaging the cell membrane, and there is no physical damage to the DNA even if it is performed for 30 seconds or more at 3,200 rpm.

The proteolytic enzyme is contained in the DNA extraction method and the DNA extraction method according to the present invention. In particular, the protease K, which has been extensively used for DNA or RNA isolation, is used at a concentration of 50 to 150 / / ml And it is most preferable that the concentration is 100 μg / ml. The step of inactivating the proteolytic enzyme in the DNA extraction method according to the present invention can be carried out by culturing at a temperature of 90 to 100 캜, preferably about 95 캜.

In the present invention, the term marine aquatic organisms refers to small flora and fauna living in marine and freshwater ecosystems, including cyprinus carpio ), carassius ( Carassius carassius ), catfish ( Silurus asotus ), loach ( Misgurnus mizolepis ), nesting species, small fish (finfish), sea urchins ( Oryzias dancena ) and green pufferfish ( Tetraodon nigroviridis ), sea anemone, flounder ( Paralichthys olivaceus ), zooplankton Artemia ( Artemia franciscana ) and phylum mollusca (class gastropoda, Reishia clavigera , Cella toreuma , ridgeline ( Patelloida saccharina lanx ), eyeballs ( Turbo coronata coreensis), Night snails (Chlorostoma lischkei), Pearl conch (Chlorostoma xanthostigma , Omphalius rusticus rusticus ), dog fence ( Monodonta labio confusa ), round mouth stubble ( Cantharidus jessoensis ), Nerita japonica , Cerithidea rhizophorarum ), beetle pox ( Cerithideopsilla cingulata ), sea bass ( Batillaria multiformis ), pods ( Batillaria cumingii , Nodilittorina radiata , Cratostoma rorifluum ), hairy pig stump ( Cantharus cecillii , Japeuthria ferrea ), black streaks ( Hima fratercula fratercula ), flower galeoptera ( Siphonaria sirius ), a tongue depressor ( Siphonaria Japonica ), the class Polyplacophora ( Acanthopleura japonica ), prosobranchia ( Haliotis discus hannai ), class Bivalvia ( Scapharca broughtonii ), a new scallop ( Scpharca subcrenata ), cockles ( Fulvia mutica ), Mactra veneriformis , Saxidomus purpurata ), baby clams ( Ruditapes bruguieri ), and microalgae include Dunaliella salina , Dunaliella tertiolecta , Tetraselmis suecica ), Phaeodactylum tricornutum , Pavova lutheri , Chlorella vulgaris , iso crysis galvana galbana ), and the like.

Hereinafter, the DNA extraction method using the tissue dissolving buffer according to the present invention will be described in detail with reference to a fish as a sample, sampling a fish tissue sample, dissolving the sample, and performing PCR analysis of the obtained DNA.

For living fish, for example, fresh tissue of about 0.4 ㎝ in width and length from the caudal fin is collected. In the method of the present invention, all the tissue samples collected from fish are applicable, but if a tissue such as eggs, scales, caudal fin, etc. is used, it is preferable that a small body can be left alive and analyzed. In addition, the DNA extraction method according to the present invention is preferably applied to a tissue collected from a fish body having a minimum total length of 2 cm.

The obtained caudal fin tissue is placed in about 100 μl of a DNA extraction buffer containing beads at a concentration of 0.5% and proteinase K at a concentration of 100 μg / ml, followed by primary culturing at 55 ° C. for 3 minutes. At this time, in order to keep the efficiency of sample crushing constant, it is preferable to bite the bottom of the tube to completely release the bead precipitate, and then add the buffer using a wide tip.

On the other hand, to shorten the total treatment time, the sample was physically disrupted using bead beating at room temperature for about 10 seconds, followed by secondary incubation at 55 ° C for 3 minutes. At this time, it is preferable that the shape of the sample is unclear and the turbidity of the buffer solution becomes high.

When the cultivation is completed, the cells are cultured for 3 minutes at a temperature of 90 to 100 ° C, preferably about 95 ° C for 1 minute to inactivate the proteolytic enzyme, centrifuged (about 13,000 rpm for 1 minute) The supernatant containing the DNA can be transferred to a new tube and used for PCR analysis by taking 1-2 μl, preferably 2 μl.

As described above, the DNA of the present invention comprising a step of culturing the tissue collected from marine aquatic organisms in a DNA extraction buffer containing a nonionic surfactant, a bead and a protease, and then inactivating the protease According to the extraction method, DNA that can be used for PCR analysis can be quickly and easily obtained within 7 minutes from the small fin tissue of living fish. In addition to the tissue samples of fish, DNA can be extracted stably and rapidly from various marine aquatic animals and plants such as plant and plankton and molluscan tissue samples and used for PCR analysis.

On the other hand, the DNA extraction method using the buffer solution for tissue dissolution according to the present invention can be applied not only to fresh tissues but also to tissue samples stored frozen and stored in alcohol for up to 3 years, The same applies to tissue samples of animal, plant plankton and mollusk animals.

In addition, the DNA solution obtained by the DNA extraction method according to the present invention exhibits the same level of PCR as immediately after the treatment even after long-term freezing, refrigeration and storage at room temperature, so that the method according to the present invention is highly stable.

According to the DNA extraction method of the present invention, it is possible to provide high quality pure DNA that can be used for PCR quickly and easily from a small amount of tissues and cells of marine aquatic organisms within a maximum of 7 minutes, And accordingly, it is possible to reduce the time required and also to process a large amount of samples, thereby improving the promptness and economical efficiency. In addition, the DNA extraction method according to the present invention can be applied not only to fresh tissues but also to tissues that have been stored for a long period of time. Since the DNA solution extracted according to the present invention can be repeatedly stored while being frozen for long periods, It is expected to contribute to securing various genetic information of living organisms.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a schematic diagram showing a process of extracting DNA of sufficient quality for PCR analysis from the tissue of the fin of a living sea otter according to the method of the present invention. Fig.
Fig. 2 is an electrophoresis image obtained by performing PCR analysis using DNA extracted from fresh caudal fin of a sea otter as a template according to the method of the present invention.
FIG. 3 is an electrophoresis image obtained by performing PCR analysis using a DNA extracted from artemia as a zooplankton as a template according to the method of the present invention.
FIG. 4 is an electrophoresis image obtained by performing PCR analysis using a DNA extracted from a suitable cell number of Dnaealella salina according to the method of the present invention as a template.
FIG. 5 is an electrophoresis image obtained by performing PCR analysis using a DNA extracted from a marine algae as a template according to the method of the present invention.
FIG. 6 is an electrophoresis image showing the result of applying the method of the present invention to a long-term stored marine biological sample.

Hereinafter, the DNA extraction method using the buffer for DNA extraction according to the present invention will be described in detail with reference to Examples. However, these examples are merely examples of the present invention, and the scope of the present invention is not limited thereto.

In the following examples, specifically,

(1) a method of extracting DNA from fish fresh fins;

(2) a method of extracting DNA from zooplankton;

(3) a method of extracting DNA from microalgae;

(4) a method of extracting DNA from mollusks; And

(5) A method for extracting DNA from tissue samples of fishes cryopreserved in alcohol for a long period of time was tested,

(6) The comparative example compares the results of the conventional marine aquatic life with the present invention and evaluates the speed and superiority of the method according to the present invention.

Example 1 : Extraction of DNA from fish's fresh fins

Fresh oyster ( O. dancena ) about 3 cm in length was stunned instantaneously using ice anesthesia, and fresh tissue about 0.4 ㎝ in length and width was collected from the caudal fin. The collected samples were washed twice with 3 times distilled water to remove water as much as possible, and then placed in a 1.5 ml microfuge tube. (10 mM Tris-Cl; pH 8.0, 50 mM KCl, 1.5 mM MgCl ₂ , 1.0% NP 40, 1.0% Tween 20) containing protease K at a concentration of 100 μg / 0.5% beads) was added to the sample. After incubation for 3 minutes in a constant temperature vessel adjusted to 55 DEG C for 3 hours to perform bead beeting for 30 seconds at room temperature and for 3 minutes at a temperature of 55 DEG C for the reaction of the tissue dissolving buffer of the present invention with the sample, Followed by tea cultivation. After the secondary culture was completed, the cells were cultured at 95 ° C. for 1 minute. The reaction solution was centrifuged (about 13,000 rpm, 1 minute), and the supernatant was transferred to a new 1.5 ml microfuge tube.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a schematic diagram showing a process of extracting DNA of sufficient quality for PCR analysis from the tissue of the fin of a living sea otter according to the method of the present invention. Fig.

In order to confirm whether or not the DNA extracted using the lysis buffer according to the present invention is abnormal in the PCR reaction, 2 μl of the supernatant was subjected to PCR under the following conditions. A pair of oligonucleotide primers (OD gb-act FW1 5'-CAGCTTGTCTCTCGCAGACA-3 '/ OD gb-act RV1 5'-AGGAAATAGGAGCCTGTGTG3') capable of amplifying a part of the cytoskeletal β- . The PCR reaction was carried out for 30 minutes at 94 ° C for 20 seconds, at 60 ° C for 20 seconds, and at 72 ° C for 20 seconds. 4 μl of the PCR product was electrophoresed on 1.5% agarose gel and observed.

The remaining components except for the beads in the buffer for DNA extraction according to the present invention were varied in concentration from 0.5 to 9 times, and about 100 μl of the composition containing protease K Was added to the sample to extract the DNA. PCR analysis was performed using the same primer pair as in Example 1 in order to confirm whether the DNA extracted by the concentration of the buffer was abnormal in the PCR reaction.

Fig. 2 is an electrophoresis image obtained by performing PCR analysis using DNA extracted from fresh caudal fin of a sea otter as a template according to the method of the present invention.

As shown in FIG. 2, PCR analysis was carried out using a DNA extracted from caudal fin obtained in the state where marine algae survived using the DNA extraction buffer of the present invention as a template, Respectively. Thus, it can be seen that the DNA obtained by the DNA extraction method of the present invention is of such a quality that the PCR reaction can be performed without any abnormality. It is also confirmed that sufficient DNA can be ensured even when the components of the buffer for DNA extraction according to the present invention are diluted or concentrated in various ways.

Example 2 : Extraction of DNA from zooplankton artemia

After washing with distilled water using A. franciscana , the water was removed as much as possible and placed in about 40 μl of the dissolution buffer of the present invention (same as that used in Example 1) prepared in a 1.5 ml microfuge tube. After primary culturing for 3 minutes in a constant temperature vessel adjusted to 55 캜, bead beeting was performed at room temperature for 30 seconds and secondary culturing was performed at a temperature of 55 캜 for 2 minutes. Then, the tertiary culture was completed at a temperature of 95 DEG C for 1 minute to obtain a supernatant containing DNA.

In order to confirm whether or not the DNA extracted using the lysis buffer of the present invention is of sufficient quality for the PCR reaction, the centrifugation supernatant was taken and a pair of primers (18s 1F 5'-TCGTCTCTTGGTGACTCTGA-3 ' / 18s 1R 5'-AAGACCGAGGTCCTATTCCA3 '). PCR was carried out by denaturing the DNA at 94 ° C for 2 minutes and repeating 30 times at 94 ° C for 30 seconds, 58 ° C for 30 seconds, and 72 ° C for 30 seconds. The PCR product was electrophoresed on 1.5% agarose gel and observed.

FIG. 3 is an electrophoresis image obtained by performing PCR analysis using a DNA extracted from artemia as a zooplankton as a template according to the method of the present invention. In FIG. 3, A is the result of PCR reaction according to the optimal incubation time required for the sample to sufficiently react with the dissolution buffer of the present invention, and B is the result of checking whether DNA extraction is possible according to the size of the sample. Here, the incubation time was the sum of the first and second incubation times. It was found that the amounts of the PCR products were similar when cultured from 5 minutes to 30 minutes. As a result, it was confirmed that the optimum incubation time for sufficient DNA extraction from artemia was 5 minutes for rapid culturing, and sufficient DNA could be obtained from a small amount of artemia tissue in the same manner as in Example 2 above. It is also confirmed that the DNA sample can be extracted sufficiently in the same manner as the tissue of 4.0 mm or more even in a trace amount of tissue of 1.0 mm or less in size.

Example 3 : Extraction of DNA from microalgae of the titration cell number

In order to confirm whether the lysis buffer according to the present invention can be used for the DNA extraction of microalgae having cell walls, the microalgae, Dunaliella salina ) were purchased and used.

After cultivation of Dulnaliella salina, the suspension was centrifuged (about 9,000 rpm, 5 minutes) to remove the medium completely by 1 × 10 ⁶ cells per 1 ml in a 1.5 ml microfuge tube. 100 μl of the lysis buffer of the present invention (the same as that used in Example 1) was added to the recovered cells, and DNA was extracted in the same manner as in Example 2.

Next, the PCR reaction was carried out using a primer pair (ITS F 5'-GGAAGGAGAAGTCGTAACAAGG-3 '/ ITS R 5'-TCCTCCCTTATTGATATGC-3') for 18s rRNA and 28s rRNA gene amplification of this species. The PCR reaction was carried out by denaturing the DNA at 94 ° C for 4 minutes, repeating 35 times at 94 ° C for 1 minute, 50 ° C for 45 seconds and 72 ° C for 2 minutes, and finally elongating at 72 ° C for 10 minutes to complete the reaction.

FIG. 4 is an electrophoresis image obtained by performing PCR analysis using a DNA extracted from a suitable cell number of Dnaealella salina according to the method of the present invention as a template. As shown in FIG. 4, the reaction time of the dissolution buffer for 1 × 10 ⁶ cells per 1 ml, which is the minimum optimal cell number for extracting DNA from microalgae, is required to be 5 minutes or longer, and the optimal incubation time is 10 minutes Respectively.

Example 4 : Reishi , a mollusk DNA extraction from clavigera

In order to confirm whether the lysis buffer of the present invention can be used for DNA extraction of marine animals, which are mollusks, some of the muscle tissues of three horseshoe crabs ( R. clavigera ) were subjected to the lysis buffer of the present invention The same procedure as in Example 2 was followed, and DNA was extracted.

Next, a primer pair (Rc-COI 1F 5'-GGTACTGCTCTAAGTCTCCT-3 '/ Rc-COI 1R 5'-AGCCAATGGAGGATACACAG-3') for amplifying the cytochrome C oxidase subunit I (COI) gene of this species was used To perform PCR reaction. The PCR was carried out by denaturing the DNA at 94 ° C for 2 minutes and repeating 30 times at 94 ° C for 20 seconds, 58 ° C for 20 seconds, and 72 ° C for 20 seconds.

FIG. 5 is an electrophoresis image obtained by performing PCR analysis using a DNA extracted from a marine algae as a template according to the method of the present invention. In FIG. 5, 1, 2, and 3 show the results of each of the perennials. As shown here, DNA was extracted from the muscle tissue of the mollusk through 100 μl of the lysis buffer of the present invention for 5 minutes, It can be seen that it was extracted.

Example 5 : DNA extraction from a sample of fish stored for a long period of time

The DNA extraction method according to the present invention was tested not only for fresh fins but also for various tissue samples of fish which were kept frozen for a long time by adding ethanol.

The samples were stored at 20 ° C. for 2 years. Three tail fin tissues and muscle tissues were taken from each of three sea cucumbers, each about 0.4 cm in width and about vertical length, and 100 μl of the dissolution buffer of the present invention was added , DNA was extracted according to Example 2 and PCR was performed.

In order to confirm whether the DNA extracts obtained according to the method of the present invention were stable for long-term cryopreservation, the supernatant samples prepared as in Example 1 were stored at 20 ° C for 2 years and PCR was carried out according to Example 1 Respectively.

FIG. 6 is an electrophoresis image showing the result of applying the method of the present invention to a long-term stored marine biological sample. In Fig. 6, A, B, D, and E were extracted from the sea tangle caudal fin tissues and muscle tissue samples, freshly hatched whole fish bodies, and oyster muscle tissue samples stored for two years in the alcohol by freezing according to the method of the present invention And C shows the PCR result after freezing the DNA extracted according to the method of the present invention for 2 years for the fresh fin sample. As shown here, both the PCR results for the long-term cryopreserved DNA extracts used as control and the PCR results for tissues frozen for up to 2 years were not significantly different in accuracy and efficiency.

Thus, it can be seen that the DNA extraction method according to the present invention can be applied not only to fresh tissues but also to tissues which have been stored frozen in alcohol for up to 2 years. In addition, it is confirmed that the DNA solution extracted according to the present invention can be cryopreserved for a long period of time and can be repeatedly used for PCR analysis.

Comparative Example : Comparison between the present invention and conventional DNA extraction method

The DNA extraction method according to the present invention was compared with the conventional method. Table 1 below shows the types of conventional DNA extraction methods used for PCR analysis of marine aquatic organisms, the kind of reagents used in the method of the present invention, the kind and amount of sample used, the number of essential extraction steps, And the time required for the operation.

Reagent type Sample type
(Volume) Extraction step After sample preparation
Total Time Way
One - Tissue dissolution buffer solution
(Tris, EDTA, SDS)
- Protease
- reagent for pure DNA separation
(Phenol, 70% ethanol,
Phenol: chloroform,
Chloroform: isoamyl alcohol,
100% ethanol or isopropanol)
-DNA solution
(H ₂ O or TE buffer solution) fin
(0.5 g or more) - tissue melting
- Organic solvent blending
- Centrifugation and supernatant recovery
-DNA precipitation
-DNA wash and drying
-DNA dissolution 12 hours
More than Way
2 - Tissue dissolution buffer solution
(Tween 20, MgCl _2, Tris, KCl)
- Protease fin
(0.5 g or more) - tissue melting
- protease inactivation
- Centrifugation and supernatant recovery 1-3 hours Way
3 - Tissue dissolution buffer solution
(NaOH, EDTA, SDS)
- neutralization solution
_{(Tris, MgCl 2, NP40,} Tween 20) fin
(0.3-0.5 g) - Tissue dissolution and neutralization
- Centrifugation and supernatant recovery Within 20 minutes Way
4 - Tissue dissolution buffer solution
(KCl, Tris, MgCl _2, Triton X-100)
- Protease Fish modified
(100) - Tissue disruption
- tissue melting
- protease inactivation
- Centrifugation and supernatant recovery 75 minutes
More than Way
5 - tissue fixative
(Ethanol or 4% formaldehyde)
- Tissue dissolution buffer solution
(Chelex 100, NaI, silica)
- Protease
- reagent for pure DNA separation
(Tris, EDTA, NaCl, ethanol)
-DNA solution
(H ₂ O or TE buffer solution) Fish scale
(4 or more) - tissue melting
- Tissue disruption
-DNA pure separation step
-DNA precipitation
-DNA wash and drying
-DNA dissolution
83 minutes
More than Way
6 - Tissue dissolution buffer solution
(Urea, Tween 20, NP 40,
Chelex 100) Pisces
Revision I
(About 602 [mu] g) - Tissue melting and shredding
- Centrifugation and supernatant recovery 15 minutes Way
7 - Tissue dissolution buffer solution
(Chelex 100 resin, TE buffer solution)
- Protease clam
musculature
(25 mg) - tissue melting
- protease inactivation
- Centrifugation and supernatant recovery 95 minutes
More than Way
8 - Tissue dissolution buffer solution
(Tris-HCl, EDTA, NACI, SDS)
- Protease
- reagent for pure DNA separation
(5M NaCl ethanol)
-TE buffer solution
-RNA degrading enzyme fin
Fish - tissue melting
-DNA pure separation step
- Centrifugation and supernatant recovery
-DNA precipitation
-DNA wash and drying
-DNA dissolution 12 hours
More than Way
9
- Tissue dissolution buffer solution
(Tris-HCl, EDTA, NaCl, SDS)
- Protease
- reagent for pure DNA separation
(Phenol, 100% ethanol,
Chloroform: isoamyl alcohol)
-DNA solution
(H ₂ O or TAE buffer solution) Adult alumina - tissue melting
-DNA pure separation step
- Centrifugation and supernatant recovery
-DNA precipitation
-DNA wash and drying
-DNA dissolution 5 hours
More than Way
10 - Tissue dissolution buffer solution
(NaOH, disodium EDTA)
- neutralization solution
(Tris-HCl) Altamia
Endurance - Tissue dissolution and neutralization
- centrifugation 35 minutes Way
11 - Tissue dissolution buffer solution
(Chelex 100) Altamia
Endurance - Tissue dissolution and neutralization 43 minutes Way
12 - Tissue dissolution buffer solution
(SDS, Tris-HCl, NaCl, EDTA)
- reagent for pure DNA separation
(Phenol: chloroform, 100% ethanol,
70% ethanol)
-DNA solution
(TE buffer solution) 1 x 10 ⁷ cells - tissue melting
- Organic solvent blending
- Centrifugation and supernatant recovery
-DNA precipitation
-DNA wash and drying
-DNA dissolution 2 hours
More than Way
13 - buffer solution for tissue dissolution 1
(Tris-Cl, EDTA, NaCl)
- buffer solution for tissue dissolution 2
(SDS, NaCl, EDTA, Tris-Cl)
- reagent for pure DNA separation
(Phenol: chloroform: isoamyl alcohol,
100% ethanol)
-DNA solution
(TE buffer solution) 3-8 × 10 ⁷ cells - tissue melting
- Organic solvent blending
- Centrifugation and supernatant recovery
-DNA precipitation
-DNA wash and drying
-DNA dissolution 34 minutes
More than example
invent - Tissue dissolution buffer solution
(Tris-Cl, KCl, MgCl ₂ , NP 40, Tween 20, beads)
- Protease Marine aquatic life - tissue melting
- protease inactivation
- Centrifugation and supernatant recovery 7 minutes
Within

As shown in Table 1, conventional DNA extraction methods have been studied extensively for tissue samples that can be obtained from fish, and it has been gradually expanded to a wide range of samples for marine animals, plants and animals . In most methods, it takes more than 60 minutes to extract DNA after sample preparation. That is, in the conventional DNA extraction method, it is expected that it takes more than 130 minutes to confirm the electrophoresis result by carrying out the PCR analysis after securing the DNA sample.

On the other hand, according to the DNA extraction method of the present invention, when the DNA extraction for various marine aquatic organisms is performed within 7 minutes, and then the PCR assay kit developed for specific marine aquatic organisms is used, It can be significantly shortened.

Meanwhile, in the case of the conventional method 6, the time required for the DNA extraction is shorter than that of other methods, instead of the time required for the DNA extraction. In this case, it is necessary to use an expensive reagent Chelex 100 resin, a high concentration of urea buffer and boiling water . Moreover, although the specific reagent of Method 6 is used to bind the protein, it may remain in the supernatant even after centrifugation, and some proteins are unable to bind depending on the acidity of the lysis buffer, and the reproducibility of the experiment And the reliability of the results can be lowered.

On the other hand, in the DNA extraction method according to the present invention, a buffer solution composed of reagents generally used in a laboratory is used. In consideration of economical efficiency and stability, a minimum suitable concentration and a culture temperature generally used in molecular biology experiments . In particular, Tris-HCl, which is a constituent component of the buffer for DNA extraction of the present invention, is a mollusk group that produces acid secretion of mucus containing lipopolysaccharides as a main component, which maintains the acidity of the buffer solution and inhibits DNA extraction This is a reagent mainly used for DNA extraction, and it is expected that mass extraction of useful genes will be possible by quickly and easily extracting pure DNA from various mollusk groups in addition to seawater and freshwater fish.

As described above, according to the DNA extraction method of the present invention, it is possible to provide high quality pure DNA which can be used for PCR quickly and easily within a maximum of 7 minutes from a small amount of tissues and cells of marine aquatic organisms, In addition to reducing the complex DNA extraction steps required by the technology and thus the time it takes to process a large amount of samples, it is possible to improve the speed and economy. In addition, the DNA extraction method according to the present invention can be applied not only to fresh tissues but also to tissues that have been stored for a long period of time. It is also possible to repeatedly confirm the gene while keeping the extracted DNA solution for a long period in a freezer, It can also be used for analysis. Accordingly, the method of the present invention can contribute to securing various genetic information of marine aquatic organisms.

Claims (9)

Tissue or cells collected from marine aquatic organisms were mixed with NP40 (Nonidet P-40; Octylphenoxy polyethoxy ethanol) and Tween 20 in a ratio of 1: 1 as a nonionic surface active agent, beads for beading beads, and protease K In a buffer solution for tissue lysis,
Bead beating the primary culture at room temperature at 1,400 to 3,200 rpm for 5 to 30 seconds,
A step of secondary culturing the bead beating-completed culture liquid,
Tertiary culture at 90 to 100 DEG C to inactivate the protease K, and
And centrifuging the third culture medium and recovering the supernatant. delete delete The method according to claim 1, wherein the bead is a glass bead having a diameter of 1.0 to 50.0 탆. The method according to claim 1, wherein the beads are contained in the buffer in an amount of 0.5 to 1.0% (v / v). The method according to claim 1, wherein protease K is contained in the buffer at a concentration of 50 to 150 占 퐂 / ml. delete (Nonidet P-40; Octylphenoxy polyethoxy ethanol) and Tween 20, a bead for beading beads and a protease K as essential components in a volume ratio of 1: 1 as a nonionic surfactant, Buffer solution composition for DNA extraction. 9. The method of claim 8, further comprising the steps of: 10 mM Tris-Cl; (pH 8.0), 50 mM KCl, 1.5 mM MgCl ₂ , 1.0% (v / v) NP 40, 1.0% (v / v) Tween 20, beads for beading and proteolytic enzyme K A buffer composition for extracting DNA from living organisms.

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