KR20100128118A - Probe and dna chip for identifying freshwater fish species - Google Patents

Abstract

PURPOSE: A method for determining classification system of inter species of fish living in fresh water is provided to simply analyze genotype of pre-larva, post-larva, young or juveniles, unmature and adult fish. CONSTITUTION: A method for determining classification of inter species of fish living in fresh water comprises: a step of isolating DNA from blood, cells or tissue of fish living in fresh water; a step of performing PCR; a step of binding the PCR product with a polynucleotide probe having identical or complementary sequence with DNA sequence selected from sequence numbers 3-18; and a step of determining classification system.

Description

Probe and DNA Chip for Identifying Freshwater Fish Species

The present invention relates to a probe and a DNA chip and kit for species determination of freshwater fish.

The present invention relates to a method for determining the species classification system of Korean freshwater fishes and related polynucleotide probes, DNA chips, and kits. In particular, the present invention relates to a simple, quick and accurate classification of Korean freshwater fishes. More specifically, to prepare a polynucleotide probe based on single nucleotide polymorphism (SNPs) present in the mitochondrial COI gene region of Korean freshwater fish and a kit comprising the same.

 There are a total of 42 species 213 and 1085 species in Korea. Among them, freshwater fish are 17 species 39 and 213 species, including 62 endemic species. 4 and 6 species of endangered wild animals and plants, 4 families and 12 species of endangered wild animals and plants II class, and 4 species of endangered wild animals and plants, and 12 species of endemic carp family (15 species), Loach family (3 species) Species), Dongja canopy (2 species), poison dog family (1 species), verbal family (1 species), Maggie family (1 species), and 12 other species introduced into Korea for aquaculture and discharge. have.

 Species classification and phylogeny of freshwater fish at home and abroad include skeletal structure, body shape, shape of caudal fin, scales, counting traits, and measurement sites of fish, including full length, body length, head length, height, dorsal fin starting distance, pectoral fin starting distance, and pear It is based on trait traits and early life history measured by measuring the straight line distance, such as fin starting distance, disease length, sickness, glasses, binocular spacing, and body width.

 Freshwater fish are divided into six stages from fertilized eggs until they become sexually active.The pre-larva stage, which is a young individual from the time of hatching until the completion of yolk absorption, and the fins from each time after the yolk absorption. The post-larva stage, a young individual until the digit becomes an integer, and the young or juveniles stage, a young entity in which the appearance of the fish after the late larvae is well distinguished from the mature ones. , Body shape, half body, body color, etc. are almost same as the adult sex, but the immature stage, the gonads are not mature, the adult sex stage (adult), the gonads are fully mature, and the gonads are closed and the spawning season An individual in spawning or off-season, with a senility stage in which body color fades or scales fall off.

 Except for the sex stage and the norm stage, not only are there many difficulties in identifying species, but even if it is not an expert, it is difficult to accurately distinguish the species. Therefore, molecular biological researches are being conducted to solve these problems, and as the genetic sequencing method has been developed, a method of identifying the species of freshwater fishes using the difference of the nucleotide sequences and revealing their flexibility relationship has been developed. In addition, freshwater fish are classified into primary freshwater fish that live their lives in pure freshwater, those that regularly flow in and out of freshwater and seawater, and those that live in the lower brackish waters affected by salinity. Depending on the spawning season, it has unique ecological habits. Especially, it is required to investigate the exact distribution of species in young populations using genetic markers.

 In order to distinguish species of Korean freshwater fish, it is necessary to accumulate sequencing data for genetic markers with high resolution because of many mutations, and there is an urgent need for an analysis method capable of quickly distinguishing nucleotide differences between species. .

 Species classification systems using common DNA are described in Tautz et al. (2002, 2003), Hebert et al. (2003) suggested that a single gene sequence can be standardized to identify most species. In fact, Mitochondria's cytochrome oxidase subunit I (COI) gene region has been shown to play an important role as a standardized biomarker for species identification. In particular, the COI gene region is more useful for identifying species because of the greater variation between species than the intra-region variation (1 ~ 2%), and the high reliability of the results has been verified. However, there is a limit in distinguishing all species with only the COI gene region, and research is being conducted to find a more efficient gene region other than COI.

To date, COIs have been recognized as standardized DNA biomarkers. COIs have been identified in database systems such as Barcode Of Life Data Systems ( www.barcodinglife.com ) and Korea Barcode Of Life ( www.koreabarcode.net ). As a standard marker, ecological and image data are accumulated along with DNA information.

 In order to solve the above problems, the present invention extracts DNA from blood, cells, and muscle tissue of Korean freshwater fish, and amplifies the PCR product of the same or complementary nucleotide sequence. It is to provide a method for analyzing the Korean freshwater fish genotype by reacting with a polynucleotide probe comprising a.

Among the freshwater fishes in Korea, the selection of inter species is 5 and 11 out of 35 species except natural monuments and endangered wild plants I and II. That is Guppy Carassius auratus (LINNAEUS, 1758), gagfish Rhodeus uyekii (MORI, 1935), the bread napjul gang Rhodeus notatus (NICHOLS, 1929), give tanakia lanceolata Acheilognathus yamatsutae (MORI, 1928), lead geographic Acheilognatus rhombeus (TEMMINCK and SCHLEGEL, 1846 ), the visible lead geographic Acanthorhodeus gracilis (REGAN, 1908) , indeed galgyeoni Zacco koreanus (KIM, OH and HOSOYA , 2005), minnow Zacco platypus (TEMMINCK and SCHLEGEL, 1902 ), kkeuri Opsariichthys uncirostris amurensis (BERG, 1940), the banks go munmang Rhinogobius giurinus (RUTTER, 1897), pushed Rhinogobius brunneus (TEMMINCK and SCHLEGEL, 1845).

 In the identification of species of freshwater fish, even if differences in sequencing are found, a standardized way to analyze them quickly and accurately can save a lot of time, human resources and costs. In addition, Korean freshwater fishes have a large number of endemic species due to the local isolation of the sea on three sides, and 17 trees 39 and 213 species, including natural monuments and endangered species, represent abundant biological diversity of the domestic freshwater ecosystem. . In the present invention, to accurately determine the difference in the nucleotide sequence of freshwater fish, and to produce a polynucleotide probe to vary according to the various on the basis of this, using a DNA chip or kid containing the same according to the species of freshwater fish It is to provide a method for quickly and accurately checking for sequence differences.

 Another object of the present invention is to provide a probe comprising 15 to 30 consecutive nucleotides, including a DNA sequence, and a kit comprising the same, including a region having a nucleotide sequence difference between species of fish as described above. . By the present invention, it is to provide a method that can easily, quickly and accurately test the genotyping of a number of Korean freshwater fish on a single slide.

 The inventors have found a monobasic polymorphic site of the COI gene in the mitochondrial DNA of various freshwater fishes, and based on this, an optimal polynucleotide probe capable of specifically binding to each species of the fish was produced.

 As described above, according to the present invention, eleven species belonging to Korean freshwater fish by extracting DNA from 11 kinds of blood, cells, and muscle tissues belonging to Korean freshwater fish, and reacting the amplified PCR products with specific polynucleotide probes. By analyzing the genotype of may provide a method for determining the classification of the fish.

 According to this classification system determination method, the genotyping analysis of 11 species belonging to a large number of Korean freshwater fishes can be easily and quickly determined on a single slide, so that the species belonging to the fish can be easily and easily determined. There is. According to the present invention, when the COI region of fish mitochondrial DNA belonging to Korean freshwater fish is used as a genetic marker, it is possible to solve the problem existing in discriminating species belonging to Korean freshwater fish. Using the COI region identified as having a large number of monobasic polymorphisms according to the present invention as a measure of analysis, it provides a more advanced type of genetic analysis method than the prior art, so that genotyping can be more effectively identified and resolution is significantly increased. You can.

 The present inventors found a monobasic polymorphic site of the COI gene among 11 mitochondrial DNAs belonging to various Korean freshwater fishes, and based on this, a polynucleotide probe capable of specifically binding to every 11 species belonging to Korean freshwater fishes was identified. I can produce it. Since the polynucleotide probe is manufactured based on 11 monobasic polymorphism sites belonging to Korean freshwater fish, the polynucleotide probe binds to the DNA of the intended fish but does not bind to the fish belonging to other species.

 After numerous studies and efforts, the present inventors have determined that the DNA sequences corresponding to SEQ ID NO: 3 to SEQ ID NO: 18 among the 11 mitochondrial DNAs belonging to Korean freshwater fish are distinguished from 11 species belonging to each Korean freshwater fish. When the polynucleotide probe having the same or complementary nucleotide sequence as the DNA sequence of SEQ ID NO: 3 to SEQ ID NO: 18 is confirmed to be optimally suitable, it is remarkably superior to other conventional methods of Korean freshwater fish. It was found that the species of the species can be identified. In addition, by using the microarray method using the above-described polynucleotide probe according to the present invention, by identifying the species-specific monobasic polymorphism according to the hybridization of DNA, 11 species belonging to Korean freshwater fish without analyzing the nucleotide sequence Has genotype and its applicability to quickly determine the species to which it belongs. In addition, it is possible to analyze the exact genotype of 11 species belonging to a large number of Korean freshwater fishes in a single experiment, and thus the applicability of technology development to standardize and automate 11 genotypes and classification system determination methods belonging to Korean freshwater fishes. Maximized. That is, the present invention significantly shortened the time required for analyzing a sample compared to the conventional method, and has an effect of processing a large amount of samples in a short time.

The present invention for achieving the above object comprises the steps of extracting DNA from the blood, cells or tissue of Korean freshwater fish; Performing a polymerase chain reaction (PCR) on the extracted DNA to obtain a PCR product; Combining the PCR product with a polynucleotide probe comprising a nucleotide sequence identical to or complementary to the DNA sequence of any one of SEQ ID NO: 3 to SEQ ID NO: 18; And determining the classification system to which the fish belongs by checking whether the combination is present.

 Another embodiment for achieving another object of the present invention is used in the method for determining the classification system of Korean freshwater fish as described above, 15 or the same as or complementary to the DNA sequence of any one of SEQ ID NO: 3 to It may be a polynucleotide probe for determining the species classification system of Korean freshwater fish, characterized in that consisting of -30 consecutive sequences and a DNA chip for determining the species classification system of Korean freshwater fish including the same.

 Another embodiment for achieving another object of the present invention may be a kit comprising the above DNA chip. That is, it binds to the DNA sequence of the nucleotide sequence of the COI gene in the mitochondrial DNA of freshwater fish of Korea, and consists of 15-30 consecutive base sequences identical or complementary to the DNA sequence of any one of SEQ ID NO: 3 to SEQ ID NO: 18. A polynucleotide probe, characterized in that; and a primer for amplifying the mitochondrial DNA of the fish by a polymerase chain reaction (PCR); kit for determining the species classification system of fish belonging to Korean freshwater fish, characterized in that it comprises a Do.

 Hereinafter, with reference to the accompanying drawings, preferred embodiments of the present invention will be described in detail.

 According to the present invention, a method for determining the species classification system of Korean freshwater fish is obtained by first extracting DNA from blood, cells, or tissues of Korean freshwater fish, and performing a PCR reaction on the extracted DNA to obtain a PCR product. Go through the steps Since the extraction of DNA from Korean freshwater fish is to determine the species to which the fish belongs by analyzing the extracted DNA, it is not particularly limited in which part of the freshwater fish is extracted or how is extracted. Among these, the extracted DNA preferably includes a monobasic polymorphism site of a cytochrome c oxidase subunit I (COI) gene region in mitochondrial DNA of freshwater fish. The PCR amplification of the extracted DNA is also performed to extend the test sample, and the method of obtaining an amplification product is not particularly limited. It is obvious that other DNA extraction methods or amplification methods known to those skilled in the art also belong to the scope of the present invention.

 The present inventors have found a single salt latogenous site of the COI gene in mitochondrial DNA of various Korean freshwater fishes, and based on this, a polynucleotide probe capable of specifically binding to each species of the fish was produced.

 1 is a schematic diagram showing the gene arrangement on the mitochondrial DNA of 11 species of fish belonging to Korean freshwater fish, the size of the mitochondrial DNA shown here is 16602bp, wherein the COI gene is located over 5486bp-7036bp .

 Subsequently, the most important feature of the present invention is to bind the PCR product with a polynucleotide probe comprising a nucleotide sequence identical or complementary to any one of SEQ ID NO: 3 to SEQ ID NO: 18. Subsequently, the step of determining whether the fish belongs to the classification system by determining whether or not the binding, this also may be used for all the combination checking method commonly used in the technical field to which the present invention belongs.

 DNA sequences of SEQ ID NO: 3 to SEQ ID NO: 18 according to the present invention are as shown in Table 1 below.

 Table 1: DNA Sequences for the Identification of 11 Species of Korean Freshwater Fishes

DNA Sequence for the Determination of Korean Freshwater Fish Species Probe name DNA sequence Reactive fish species DNA sequence position A1 (SEQ ID NO: 3) 5'-TAACCGCCGTCCTCCTTCTCCTA-3 ' crucian carp 524-546 A2 (SEQ ID NO: 4) 5'-CTTCTATCGCTACCCGTTCTGGC-3 ' Rope 541-563 A3 (SEQ ID NO: 5) 5'-CTTCTCCTGTCCTTACCCGTACT-3 ' Pyramid 538-560 A4 (SEQ ID NO: 6) 5'-CACCCTTATTCGTCTGAGCCGTA-3 ' Pyramid 497-519 A5 (SEQ ID NO: 7) 5'-CCTTTTTGTATGAGCTGTACTTG-3 ' Crab 501-523 A6 (SEQ ID NO: 8) 5'-CAGACCGCAACTTAAACACAACC-3 ' Craving 587-609 A7 (SEQ ID NO: 9) 5'-GAGCCGTTCTAATTACAGCTGTC-3 ' Craving 512-534 A8 (SEQ ID NO: 10) 5'-CTTTCCCTTCCCGTTCTTGCCG-3 ' Push 544-565 A9 (SEQ ID NO: 11) 5'-AATAGTAGGTACGGCCCTTAGCC-3 ' Push 34-56 A10 (SEQ ID NO: 12) 5'-CTGAACTTAGCCAGCCAGGCTCA-3 ' Rice cake 69-91 A11 (SEQ ID NO: 13) 5'-GCTATCTTTACCAGTCTTGGCCG-3 ' Hornfish 543-565 A12 (SEQ ID NO: 14) 5'-CCGGGATTACGATGCTCCTTACG-3 ' Chill 566-588 A13 (SEQ ID NO: 15) 5'-CTATCCCTACCCGTATTGGCTGC-3 ' Chill 544-566 A14 (SEQ ID NO: 16) 5'-GTACTTCTCCTTTTATCTCTACC-3 ' Thorn Lead Geography 532-554 A15 (SEQ ID NO: 17) 5'-GTACTTCTACTGCTATCACTACC-3 ' Geography 532-554 A16 (SEQ ID NO: 18) 5'-AACACCTTTATTCGTATGAGCTG-3 ' Geography 495-517

 The DNA sequence according to the present invention as shown in Table 1 above is to determine the classification system of freshwater fish from Korea, and to determine which species of the freshwater fish from Korea. The DNA sequence includes a monobasic polymorphism site of the COI gene in mitochondrial DNA of freshwater fish of Korea, and represents a part of a nucleotide sequence that can be classified according to the fish species of the fish. The location of each DNA sequence is also shown. Therefore, the polynucleotide probe according to the present invention preferably has a nucleotide sequence complementary to the DNA sequence so as to bind to the DNA sequence of SEQ ID NO: 3 to SEQ ID NO: 18. In addition, in using a PCR product obtained by amplifying the DNA extracted from the fish, it is possible to use either strand of the PCR product, which has a base sequence complementary to the DNA sequence of SEQ ID NO: 3 to SEQ ID NO: 18 In order to react with the target PCR product, the probe according to the present invention may have the same base sequence as the DNA sequence of SEQ ID NO: 3 to SEQ ID NO: 18. The present inventors used the base sequence complementary to the DNA sequence of the PCR product according to each sequence number, and named each probe name corresponding to the sequence number A1 to A16.

 2 to 6 are schematic views showing 630 sequential sequences including a single nucleotide polymorphism site of the COI gene in mitochondrial DNA of Korean freshwater fish according to a preferred embodiment of the present invention. That is, Figure 2 is a variety of Korean freshwater fish, in particular, Carassius auratus, horneye (Rhodeus uyekii), Rhodeus notatus, Acheilognathus yamatsutae, Acheilognatus rhombeus, spiny lead COI genes from the mitochondrial DNA from the species of Acanthorhodeus gracilis, Zacco koreanus, Zacco platypus, Opsariichthys uncirostris amurensis, Rhinogobius giurinus, Rhinogobius brunneus DNA sequence of up to 150, Figure 3 is a DNA sequence of 151 to 300 of the gene, Figure 4 is a DNA sequence of No. 301 to 450 of the gene, Figure 5 is 451 of the gene To 600 DNA sequence, Figure 6 shows the DNA sequence from 601 to 636 of the gene sequentially.

 Here, the monobasic polymorphism site refers to a nucleotide sequence showing a genotype different from 11 nucleotide sequences belonging to Korean freshwater fish, as shown in FIGS. That is, the site indicated by ". (Dot)" indicates the same genotype as the nucleotide sequence corresponding to the crucian carp, the specific A, C, G or T is the site showing monobasic polymorphism.

 2 to 6 are sequences of about 636 nucleotide sequences including a single nucleotide polymorphism site of a cytochrome c oxidase subunit I (COI) gene among 11 mitochondrial DNAs belonging to Korean freshwater fish according to a preferred embodiment of the present invention. It is a schematic diagram as shown. This link is a comparison of 636 sequences of 11 mitochondrial DNA COI regions belonging to Korean freshwater fish, showing that there are a number of unique monobasic polymorphisms in a particular fish species. Although shown in 50 nucleotide sequence units on the limit of the page, Figures 2 to 6 mean the drawings in order. Among them, the location region of the DNA sequence shown in Table 1 is a region where species-specific monobasic polymorphism exists that can be used as a polynucleotide probe to be used in the microarray method according to the present invention.

 After numerous studies and efforts, the inventors found that the DNA sequence corresponding to SEQ ID NO: 3 to SEQ ID NO: 18 in the COI gene among 11 mitochondrial DNAs belonging to Korean freshwater fish is optimally suitable for distinguishing the species of each fish. Using the polynucleotide probe having the same or complementary nucleotide sequence as SEQ ID NO: 3 to SEQ ID NO: 18, it is possible to determine the classification of 11 species belonging to freshwater fish of Korea significantly superior to any other conventional method It was confirmed that there is. Since these polynucleotide probes are manufactured based on the monobasic polymorphism sites of the fish species described above, it is preferable that the polynucleotide probe binds to DNA of fish belonging to the intended reactive fish species but not to those of fish belonging to other species. .

 As used herein, 'polymorphism' refers to the generation of two or more alternative sequences or alleles within a genetically determined population. The polymorphic marker or site is the location where divergence occurs. Preferred markers have two or more alleles that exhibit a frequency of occurrence of at least 1%, more preferably at least 10% or 20%, in the selected population. The polymorphic site may be a single base pair. Accordingly, the present invention may be a nucleotide or nucleic acid fragment comprising the same or a complement thereof including one or more polymorphic sites according to the present invention in addition to the above-described nucleotide sequence. In addition, the polymorphic site according to the present invention means that two or more alternative sequences or alleles occur in a genetically determined population, as described above, and is identical to the nucleotide sequence of the polymorphic site shown in FIGS. 2 to 6. Or a base sequence including a DNA sequence capable of complementarily binding thereto.

 Based on such polymorphic sites, the polynucleotide probe according to the present invention corresponds to the present invention as long as the polynucleotide probe includes a nucleotide sequence capable of complementarily binding to the DNA sequence of any one of SEQ ID NO: 3 to SEQ ID NO: 18. do. Preferably, at least 10 and up to 100 consecutive polynucleotides or complements thereof, including base sequences capable of complementarily binding to the DNA sequence, more preferably 10 or more and 50 It is the following and most preferably, it may be 15 or more and 30 or less.

 The polynucleotide probe is characterized by a monobasic polymorphism site in the COI gene region in the mitochondrial DNA of the fish in binding to a target DNA extracted from 11 species belonging to Korean freshwater fish or a PCR product amplified the DNA. The combination is characterized in that it is made different depending on the classification system to which the fish belongs, that is, 11 species belonging to Korean freshwater fish. Thus, the present invention determines the species of the fish according to the combination of the probe and the target product, the species of the fish that can be analyzed by the probe according to the present invention is as described in the reaction species of Table 1 above.

 Reaction fish species in Table 1 refers to a kind of Korean freshwater fish that can be characteristically bound to the DNA sequence according to the present invention. In other words, the DNA base sequence corresponding to SEQ ID NO: 3 to 18 in Table 1 binds to the DNA extract of the reactive fish species listed in Table 1, but does not bind to other fish species other than the above-mentioned reactive fish species. Therefore, in the present invention, when the polynucleotide probe of any one of SEQ ID NO: 3 to SEQ ID NO: 18 may be characterized in that to determine the species to which the fish belong.

 Specifically, the binding is different depending on the classification system to which the fish belongs, as shown in Table 1, the polynucleotide probe of SEQ ID NO: 3 (this includes a base sequence complementary to the DNA sequence of SEQ ID NO: 3) Means the polynucleotide probe, which is the same in each sequence number below) specifically binds to the 524-546th DNA sequence of the COI gene region in the mitochondrial DNA of Carassius auratus species, or in the case of SEQ ID NO: 4 Is the 541-563 DNA sequence of Acheilognathus yamatsutae species, SEQ ID NO: 5 to SEQ ID NO: 6 for the 538-560, 497-519 DNA sequence, SEQ ID NO: 7 of the pyramid (Zacco platypus) species Is 501-523 DNA sequence of Zacco koreanus species, 587-609th, 512-534 DNA sequence of Rhinogobius giurinus species in SEQ ID NO: 8 to SEQ ID NO: 9, SEQ ID NO: 10 to SEQ ID NO: 11 is the 544-565, 34-56 DNA sequence of the Hingobius brunneus species, SEQ ID NO: 69-91 DNA sequence of the Rhodeus notatus species In the case of SEQ ID NO: 13, the 543-565th DNA sequence of Rhodeus uyekii species, and in the case of SEQ ID NO: 14 to 15, the 566-588th and 544-566 DNA of Opsariichthys uncirostris amurensis species SEQ ID NO: 16 for the Acanthorhodeus gracilis species, 532-554 DNA sequence; for SEQ ID NO: 17 to SEQ ID NO: 18 for the 532-554th species of Acheilognatus rhombeus species, 495-517 And specifically bind to the first DNA sequence.

 As described above, the present invention uses a DNA sequence of any one of SEQ ID NO: 3 to SEQ ID NO: 18 having a single nucleotide polymorphism site in species belonging to various Korean freshwater fish, the same as or complementary to the DNA sequence The polynucleotide probe, which contains a sequence that can be specifically bound, is combined with the DNA extract (exactly the PCR product) of the fish we want to know. As such, the polynucleotide probe according to the present invention is manufactured based on each of the 11 mononucleotide polymorphism sites belonging to Korean freshwater fish, and binds to DNA of fish corresponding to the intended fish species, but belongs to other species. Since it is characterized in that it does not bind, it is preferable that the step of binding the polynucleotide probe is performed at least one or more times. Accordingly, if the binding of the extracted DNA and the probe according to the present invention is repeatedly performed before the step of confirming the binding, the binding process can be more surely established, thereby further solidifying the binding of the probe and the target DNA product. In addition, after the step of confirming the binding, repeatedly performing the combination of the extracted DNA and the probe according to the present invention has the effect of predicting the overlapping determination using a variety of probes. Therefore, the step of binding the polynucleotide probe is preferably performed repeatedly at least one or more times a number of times (particularly 3 to 4 times).

 Another embodiment of the present invention is a step of extracting DNA from 11 kinds of blood, cells or tissues belonging to Korean freshwater fish, and performing a polymerase chain reaction (PCR) on the extracted DNA to obtain a PCR product In the polymerase chain reaction (PCR), a polynucleotide consisting of a DNA sequence of SEQ ID NO: 1 or a polynucleotide consisting of a base sequence complementary to the DNA sequence is used as a forward primer, or the DNA of SEQ ID NO: 2 It may be characterized by using a polynucleotide consisting of a sequence or a polynucleotide consisting of a base sequence complementary to the DNA sequence as a reverse primer.

 In other words, a specific primer is used to amplify DNA extracted from 11 species belonging to Korean freshwater fish. This specific primer corresponds to the mononucleotide polymorphism DNA sequence of the COI gene in the mitochondrial DNA of the fish, thereby further amplifying the extracted DNA. To this end, the DNA extracted from blood, cells or tissues as described above preferably comprises a monobasic polymorphism site of the COI gene.

 The forward primer and the reverse primer used to amplify the DNA extracted in the present invention by PCR are as shown in Table 2 below.

 [Table 2: Primer for discriminating 11 kinds of fish belonging to Korean freshwater fish]

primer Sequence location Forward primer (SEQ ID NO: 1) 5’-GGTCAACAAATCATAAAGATATTGG-3 ’ 5508-5533bp Reverse primer (SEQ ID NO: 2) 5’-TAAACTTCAGGGTGACCAAAAAATCA-3 ’ 6194-6220bp

 As shown in Table 2, the primer used in the PCR process of the present invention is also formed to be optimally suitable for the DNA sequence of the monobasic polymorphism site of the COI gene in mitochondrial DNA of freshwater fish from Korea. Here, the forward primer is to bind to the 5508-5533bp region of the COI gene located over 5486bp-7036bp in the mitochondrial DNA of freshwater fish from Korea, the reverse primer is bound to the 6194-6218bp region of the COI gene. It is based on the most common sequencing, regardless of the various fish species. After investigating and analyzing DNA sequences and SNPs of various fish species shown in FIGS. 2 to 6, the present inventors can most effectively produce PCR products when using the nucleotide sequences consisting of SEQ ID NO: 1 and SEQ ID NO: 2 above. It was confirmed that there is. In other words, regardless of the various fish species mitochondrial DNA extract will be amplified by the PCR method, thereby increasing the number of PCR products amplified significantly.

 In addition, the present invention is to confirm the hybridization reaction after the PCR product and the probe according to the present invention, Rhodamine (rhodamine), cy3, cy5 or biotin is attached to the forward primer or reverse primer In order to determine whether the binding is performed, a species discrimination method of 11 kinds of fish belonging to the Korean freshwater fish, which is characterized in that the binding by detecting the fluorescence expressed by the rhodamine, cy3, cy5 or biotin is also possible. . Specifically, the confirming step may be characterized in that genotyping is conveniently performed by using rhodamine, cy3, cy5 or Syreptavidin-Cyanine in combination with biotin, and analyzing the results using a microarray scanner. have.

 Another embodiment for achieving the other object of the present invention is to be used in the method for determining the classification system of freshwater fish from Korea as described above, 15 or the same as or complementary to the DNA sequence of any one of SEQ ID NO: 3 to It is a polynucleotide probe for determining 11 classification systems belonging to Korean freshwater fish, characterized by consisting of -30 consecutive nucleotide sequences. Furthermore, the present invention binds to the mononucleotide polymorphism DNA sequence of the COI gene in 11 mitochondrial DNAs belonging to Korean freshwater fish, and is identical to or complementary to the DNA sequence of any one of SEQ ID NOs: 3 to 18. It may be a DNA chip for determining the taxonomy of 11 species belonging to the Korean freshwater fish including a polynucleotide probe characterized in that consisting of 30 consecutive nucleotide sequences. Here, the polynucleotide probe is preferably in the form of being immobilized on a substrate on the glass.

 In the present invention, by using DNA microarray technology, it is possible to simultaneously determine 11 classification systems belonging to multiple Korean freshwater fishes on one slide. Eighteen probes capable of determining the species belonging to the fish were integrated by setting a plurality of zones on a glass slide treated with a chemical functional group, and the targets were labeled with a fluorescent material label obtained by symmetric or asymmetric PCR. By confirming the hybridization reaction of the gene sequence, it is possible to distinguish exactly 11 fish.

 As described above, the polynucleotide probe is composed of 15-30 consecutive base sequences identical or complementary to any one of SEQ ID NO: 3 to SEQ ID NO: 18. Because it is manufactured on the basis of, it is characterized by binding to the target product DNA of the intended fish but not to other fish. Accordingly, by using the polynucleotide probe having the same or complementary nucleotide sequence as SEQ ID NO: 3 to SEQ ID NO: 18, it is possible to distinguish 11 species belonging to Korean freshwater fish remarkably superior to any other conventional methods. .

 In addition, another embodiment for achieving another object of the present invention is a kit for determining 11 classification systems belonging to the Korean freshwater fishes used in the 11 classification system determination method belonging to the Korean freshwater fish as described above Can be.

 That is, it binds to the mononucleotide polymorphism DNA sequence of the COI gene among 11 mitochondrial DNAs belonging to Korean freshwater fish, and is 15-30 consecutively identical or complementary to the DNA sequence of any one of SEQ ID NOs: 3 to 18. A polynucleotide probe comprising a nucleotide sequence; and a primer for amplifying the mitochondrial DNA of the fish by a polymerase chain reaction (PCR); 11 classification of Korean freshwater fish belonging to It may be a kit.

 The kit for determining the classification system is a genotype of a fish to be analyzed without analyzing the sequencing by discriminating species-specific monobasic polymorphism according to hybridization of DNA by using the microarray method using the polynucleotide probe described above. It has the utility to quickly determine the family and its clan. In addition, it is possible to analyze precise genotypes of multiple fish species belonging to Korean freshwater fishes in one experiment, thereby maximizing the applicability of technology development to standardize and automate the method of determining the genotype and classification system of the fishes. . That is, the present invention significantly shortened the time required for analyzing a sample compared to the conventional method, and has an effect of processing a large amount of samples in a short time.

 Hereinafter, one preferred embodiment of the present invention will be described in detail with reference to the accompanying drawings. The invention may be better understood by the following examples, which are intended for purposes of illustration of the invention and are not intended to limit the scope of protection defined by the appended claims.

 Example 1: Primer Preparation for Amplification of Extracted DNA

 Primers for amplifying DNA extracted from various Korean freshwater fish were synthesized. Primers used in the PCR method according to the invention are as shown in Table 1 above. Primers used in the present invention was prepared by the request of BIONEER of Korea.

 Example 2: Probe Preparation According to Fish Species

 Probes were synthesized to identify specific variations of Korean freshwater fish. Sequence information of the probe used in the hybridization reaction in the present invention is as shown in Table 1 above. In the present invention, an aldehyde functional group is processed to prepare a probe as a DNA chip, which modifies an amino link at the 5 'end of the selected probe to covalently bond the probe according to the present invention on a glass, and then, on the glass slide during the hybridization reaction. In order to minimize spatial interference between integrated probes, 10-20 oligos (dT) were added and synthesized. The probe used in the present invention was prepared by requesting a BIONEER company in Korea.

 Example 3 DNA Extraction and PCR Reactions

 1) The 11 kinds of DNA belonging to Korean freshwater fish were extracted using DNIA's DNeasy tissue kit from QIAGEN, Germany.

 2) PCR reaction was performed in a DNA engine (MJ Research, USA) by the method of the conditions shown in Table 3 below.

 3) After PCR, 1ul of gel loading buffer (0.2% orange G, 0.25% xylene cyanol FF, 60% glycerol) was added to 5ul of PCR product and electrophoresed on 1.5% agarose gel containing 1ug / ml ethidium bromide. Afterwards, the PCR band was confirmed by an image analyzer (DNr Bio-Imaging Systems, China) with a UV transilluminator.

TABLE 3: Primer Normal Amplification Confirmation Conditions

Reaction composition Reaction conditions 5X buffer 5ul 94 ℃, 5 minutes 1 Cycle dNTP 1ul Potential primer 1ul 94 ° C, 30 seconds 30 Cycle Inversion primer 1ul 50 ° C, 1 minute Taq 0.3ul 72 ° C., 1 minute 30 seconds DW 14.7ul 72 ° C., 10 minutes 1 Cycle Template 2ul

 Example 4 Fabrication of Chips for Discrimination

 1) The same amount of 2X spotting buffer (6X) as the probe prepared according to Example 2 wherein the amino link was modified at a concentration of 50 uM on a CSS-100 Silylated Slide (Cell Associate, USA) to fabricate the oligonucleotide chip. SSC, 3M Betaine) was mixed and integrated, which was reacted at room temperature for 16 hours. After the reaction was completed, the slide was washed once with 0.1% SDS for 5 minutes, and then twice with distilled water for 5 minutes.

 2) After reacting the chip made in sodium borohydride (1.3g NaBH4 375ml, PBS 125ml, 100% EtOH) for 5 minutes, washed twice with distilled water for 5 minutes, and dried for 5 minutes at 800 rpm in a vacuum centrifuge It was.

 3) The samples were separated in a perfusion chamber (BioGrace, USA) for multiple reactions on a single slide and stored in the dark at room temperature until the experiment.

 The DNA chip thus produced is exemplarily shown in FIG. 7. FIG. 7 is a schematic diagram illustrating the structure of a DNA chip including an oligonucleotide probe prepared based on the monobasic polymorphism shown in FIGS. 2 to 6.

Example 5 Hybridization Reaction

 1) PCR products bound with rhodamine, cy3, cy5 or biotin were mixed with hybridization solution (3X SSC, 0.3% Sarcosyl) in a ratio of 1: 9. When biotin was used to generate fluorescence, Syreptavidin-Cyanine was added at a concentration of 1 ug / ml and reacted.

 2) The prepared hybridization solution was applied to the chmaber and reacted at 55 degrees for 1 hour.

 3) The first wash for 5 minutes in 1X SSC, 0.1% Sarcosyl and then twice for 3 minutes in 0.1X SSC.

 4) The washed slides were dried for 5 minutes at a speed of 800rpm in a centrifuge.

 5) Confirmation of the experimental results was performed using a GenePix 4000B (Axon Instrument, USA) scanner to measure the fluorescence genotype was analyzed, the results are shown in Figures 8a to 8k.

 8A to 8K are photographs showing the reaction results after hybridizing the oligonucleotide probe and 11 PCR amplification products belonging to various Korean freshwater fishes according to the present invention using the DNA chip of FIG. 7, wherein FIG. 8A Is a crucian carp, FIG. 8b is a rope sack, FIG. 8c is a pyramid, FIG. 8d is a cramped fish, FIG. 8e is a crampfish, FIG. 8f is a push, FIG. 8g is an oak, FIG. FIG. 8J shows the results for the visible geography and FIG. 8K for the lead geography. As shown here, according to the present invention, since the fluorescent label is different depending on the type of 11 species belonging to the Korean freshwater fish, it was confirmed that the 11 species belonging to the Korean freshwater fish can be discriminated accordingly.

 On the other hand, while the present invention has been shown and described with respect to specific preferred embodiments, the present invention is variously modified and modified without departing from the technical features and fields of the present invention provided by the claims below It will be apparent to one of ordinary skill in the art that the change can be made.

 1 is a schematic diagram showing the gene sequence of 11 mitochondrial DNA belonging to Korean freshwater fish,

 2 to 6 are sequences of about 630 nucleotide sequences including a single nucleotide polymorphism site of a cytochrome c oxidase subunit I (COI) gene among 11 mitochondrial DNAs belonging to Korean freshwater fish according to a preferred embodiment of the present invention. It is a schematic diagram shown by

 FIG. 7 is a schematic diagram illustrating the structure of a DNA chip including an oligonucleotide probe prepared based on the monobasic polymorphism shown in FIGS.

 8A to 8K are photographs showing the reaction results after combining the oligonucleotide probes according to the present invention and 11 PCR amplification products belonging to various Korean freshwater fishes using the DNA chip of FIG. 7.

Claims (11)

Extracting DNA from blood, cells, or tissue of Korean freshwater fish; Performing a polymerase chain reaction (PCR) on the extracted DNA to obtain a PCR product; Combining the PCR product with a polynucleotide probe comprising a nucleotide sequence identical to or complementary to the DNA sequence of any one of SEQ ID NO: 3 to SEQ ID NO: 18; And determining the classification system to which the fish belong by checking whether the combination is present. 2. The classification system of freshwater fish according to claim 1, wherein the extracted DNA includes single nucleotide polymorphisms (SNPs) of a cytochrome c oxidase subunit I (COI) gene region of mitochondrial DNA of the freshwater fish. How to decide. The method of claim 1, wherein the polynucleotide probe comprises 15-30 consecutive sequencing sequences. According to claim 1, wherein the polynucleotide probe comprising a nucleotide sequence complementary to any one of the DNA sequence of SEQ ID NO: 3 to 18 binds to the monobasic polymorphism site of the COI gene region in the mitochondrial DNA of the fish, Wherein the combination is different according to the classification system to which the fish belongs to the classification method of freshwater fish. The method according to claim 4, wherein the binding is made differently according to the classification system to which the freshwater fish belongs, wherein the polynucleotide probe of SEQ ID NO: 3 (which comprises a base sequence complementary to the DNA sequence of SEQ ID NO: 3) Meaning the same in each of the following SEQ ID NOs) specifically binds to the 524-546th DNA sequence of the COI gene region of the mitochondrial DNA of the Carassius auratus species, or in the case of SEQ ID NO: 4, Acheilognathus yamatsutae) 541-563 DNA sequence, SEQ ID NO: 5 to SEQ ID NO: 6 for the 538-560, 497-519 DNA sequence of the pyramid (Zacco platypus) species, Zangco for SEQ ID NO: 7 koreanus) 501-523 DNA sequence, SEQ ID NO: 8 to SEQ ID NO: 9 in the case of Rhinogobius giurinus species 587-609, 512-534 DNA sequence, SEQ ID NO: 10 to SEQ ID NO: 11 is the 544-565th, 34-56th DNA sequence of Rhinogobius brunneus species, 69-91th DNA sequence of the Rhodeus notatus species is SEQ ID NO: 13 Is the 543-565th DNA sequence of Rhodeus uyekii species, 566-588th, 544-566th DNA sequence of SEQ ID NO: 16 for SEQ ID NO: 14 to SEQ ID NO: 15 The DNA sequence of the Acanthorhodeus gracilis species is the 532-554th DNA sequence, and SEQ ID NO: 17 to SEQ ID NO: 18 is specific to the 532-554, 495-517 DNA sequence of the Acheilognatus rhombeus species Method for determining the classification system of freshwater fish, characterized in that the combination. The method of claim 1, wherein the binding of the polynucleotide probe is performed at least one time. According to claim 1, wherein the polymerase chain reaction (PCR) is carried out using a polynucleotide consisting of a DNA sequence of SEQ ID NO: 1 or a polynucleotide consisting of a base sequence complementary to the DNA sequence as a forward primer, or SEQ ID NO: A polynucleotide consisting of a DNA sequence of 2 or a polynucleotide consisting of a nucleotide sequence complementary to the DNA sequence is used as a reverse primer. The method of claim 7, wherein the forward primer or the reverse primer is attached to the rhodamine (rhodamine), cy3, cy5 or biotin, and confirming whether the binding is fluorescence expressed by the rhodamine, cy3, cy5 or biotin Determination method of the classification system of freshwater fish, characterized in that for detecting whether or not combined. 15-30 consecutive bases that are the same or complementary to the DNA sequence of any one of SEQ ID NO: 3 to SEQ ID NO: 18 for use in the method for determining the classification system of freshwater fish according to any one of claims 1 to 8. Polynucleotide probe for determining the classification system of freshwater fish, characterized in that consisting of a sequence. Binding to the mononucleotide polymorphism (SNP) region DNA sequence of the COI gene in mitochondrial DNA of freshwater fish, consisting of 15-30 consecutive nucleotide sequences identical or complementary to the DNA sequence of any one of SEQ ID NOs: 3 to 18 DNA chip for determining the classification system of freshwater fish comprising a polynucleotide probe. Binding to the mononucleotide polymorphism (SNP) region DNA sequence of the COI gene in mitochondrial DNA of freshwater fish, consisting of 15-30 contiguous sequences identical or complementary to the DNA sequence of any one of SEQ ID NO: 3 to SEQ ID NO: 18 A polynucleotide probe, characterized in that; And a primer for amplifying mitochondrial DNA of the freshwater fish by a polymerase chain reaction (PCR).

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