KR100957828B1 - A new primer set and a method of selecting an hanwoo individual by using the said primer set - Google Patents

Abstract

The present invention relates to a novel primer set and a method for identifying individual cows using the same, and more particularly, whether or not a variety of beef in the market corresponds to a specific Hanwoo, that is, whether or not individual cows can be identified quickly and easily. In addition, it provides a novel primer set for PCR with high power, a method for identifying Korean cattle, and an identification kit using the primers.

Hanwoo, individual identification, primer, PCR

Description

A new primer set and a method for identifying a Hanwoo individual using it {A NEW PRIMER SET AND A METHOD OF SELECTING AN HANWOO INDIVIDUAL BY USING THE SAID PRIMER SET}

The present invention relates to a novel primer set and a method for identifying a Korean cattle individual using the same.

In recent years, the consumption of meat has increased as the Korean economy has been rapidly westernized along with the growth of the national economy and the increase of income. Beef demand has also increased. Meanwhile, as the beef market is fully opened, imported beef enters the domestic market indiscriminately, and imported beef, which is relatively cheaper than Korean beef, is quickly dominating the domestic beef market.

Domestic livestock farmers' response strategy is to differentiate them from imported beef through the production of high quality, high quality beef. The high quality of Korean beef has deepened the price gap between Korean beef and imported beef, and the price gap caused frequent cases of distributional irregularities in which cheap imported beef was sold as expensive Korean beef. For example, several cases related to the sale of mud-carps, such as the sale of mixed beef and imported meat with Hanwoo beef, were made at department stores and department stores, including Hanwoo meat gift sets.

In addition, with the recent improvement of the economic level, interest in health is increasing day by day, and in particular, the consumer's eye level on the safety of foods directly connected to health is increasing. In particular, the incidents and incidents related to beef that have occurred in recent decades have been sufficient for consumers to question the stability of beef, and consumers want to be able to accurately and transparently transmit various information about commercially available beef.

The tag used to identify Korean cattle is currently attached at the time of birth, but it is lost during the distribution process to slaughterhouses, processing plants, stores and restaurants after birth. It is causing a problem. In the case of various diseases occurring in cows due to the loss of such information and serious problems in foods using beef, there are many difficulties in determining the cause and preparing quick measures.

Therefore, in case of problems related to the safe supply of beef, it is possible to quickly identify the cause and prepare the countermeasures, and to secure the competitiveness of Korean beef, to distinguish between Korean beef and red meat or imported meat, and to accurately identify varieties or individuals of Korean beef. There is an urgent need for the development of such techniques.

A method of recent interest in this regard is the traceability system through identification of individuals at the DNA level.

Research materials for individual identification in cattle are mainly microsatellite DNAs and single nucleotide polymorphisms (SNPs), and it is confirmed that high accuracy can be achieved in experiments using them.

Specific examples include individual identification methods using 32 SNP markers found in 18 autosomes and 2 sex chromosomes (Heaton MP, Harhay GP, Bennett GL, Stone RT, Grosse WM, Casas E, Keele). . JW, Smith TP, Chitko- McKown CG, Laegreid WW.Selection and use of SNP markers for animal identification and paternity analysis in US beef cattle Mamm Genome 13 (5): 272-81, and the (2002)), this American Angus Estimates of allele and genotype frequencies applied to the cattle and other breeds showed that the probability of being identified as the same individual was 1.9 X 10 ^-10 and 2.0 X 10 ^-13 , respectively. It is reported to have an accuracy of 99.4%.

In addition, a study on the identification of individual cattle using microsatellite DNA markers for the polymorphism analysis of cattle suggested by Applied Biosystems, StockmarkerTM, ISAG and Roslin Research Institute Analysis and selection of microsatellite that can be used for the production history of Korean cattle, Journal of Animal Science and Technology 47 (4): 491-500 (2005)).

However, although the individual identification method using the microsatellite DNA marker and the SNP marker is known to exhibit high reliability in accuracy and reproducibility, it requires expensive equipment for analysis and requires expert personnel for equipment operation and analysis. There is a limit that can not be utilized quickly in the field, such as.

Therefore, there is a need for the development of a traceability system that can identify individuals at the DNA level simply and quickly without a high degree of expertise in a cheaper way.

In order to solve the problems of the prior art, the present invention is a novel primer set that can quickly and accurately identify the individual of the cattle, while securing a level of power that can be used in the Hanwoo production history tracking system, Hanwoo individual using the same An object of the present invention is to provide an identification method and a detection kit for identifying Korean cattle.

In order to achieve the above object, the present invention provides a primer set for identifying a Korean cattle individual.

In addition, the present invention provides a method for identifying a Korean cattle individual using the primer set for identifying Korean cattle individual.

In addition, an object of the present invention is to provide a detection kit for identifying Korean cattle using the Hanwoo individual identification primer set.

As a result of comparing and analyzing the DNA sequence of a cow and the DNA sequence of a foreign cow, the present inventors can identify and search for an indel, insertion and deletion region and a mismatch site unique to the cattle. A primer set was prepared, and the result of analyzing the amplified product amplified by the sample DNA using the same, and confirmed that it was possible to identify the individual of the cattle, developed the method of identifying the individual of the cattle, the present invention was completed.

Hereinafter, the present invention will be described in detail.

In the present invention, Hanwoo (Hanwoo) is a unique prop species of Korea for thousands of years to adapt to the Korean climate for a suitable constitution and refers to livestock that are reared (in one) or for breeding for meat.

In the present invention, an indel, insertion and deletion region and mismatch region inherent in Korean cattle are compared or searched with genome data of foreign cattle, and the insertion or deletion that appears to be predominantly specific to Korean cattle or Korean cattle. A site or mismatch site.

The present invention provides a primer set for identifying a Korean cattle individual.

In detail, the primer set for identifying a Korean cattle individual may amplify a nucleotide sequence including a region capable of identifying an indel, Insertion and Deletion, or mismatch region unique to Korean cattle. By identifying the size, number, and annealing temperature (T _A ) of the amplified product amplified using the primer set, that is, the temperature at which the double strand of DNA is separated into one strand, the individual of the cattle can be distinguished. Means a set of primers.

The primer set may include a first primer set including a primer comprising a nucleotide sequence of SEQ ID NO: 1 and a primer comprising a nucleotide sequence of SEQ ID NO: 2; A second primer set comprising a primer comprising a nucleotide sequence of SEQ ID NO: 3 and a primer comprising a nucleotide sequence of SEQ ID NO: 4; A third primer set consisting of a primer comprising a nucleotide sequence of SEQ ID NO: 5 and a primer comprising a nucleotide sequence of SEQ ID NO: 6; A fourth primer set consisting of a primer having a nucleotide sequence of SEQ ID NO: 7 and a primer having a nucleotide sequence of SEQ ID NO: 8; A fifth primer set consisting of a primer having a nucleotide sequence of SEQ ID NO: 9 and a primer having a nucleotide sequence of SEQ ID NO: 10; A sixth primer set consisting of a primer having a nucleotide sequence of SEQ ID NO: 11 and a primer having a nucleotide sequence of SEQ ID NO: 12; A seventh primer set consisting of a primer having a nucleotide sequence of SEQ ID NO: 13 and a primer having a nucleotide sequence of SEQ ID NO: 14; An eighth primer set consisting of a primer having a nucleotide sequence of SEQ ID NO: 15 and a primer having a nucleotide sequence of SEQ ID NO: 16; A ninth primer set comprising a primer having a nucleotide sequence of SEQ ID NO: 17 and a primer having a nucleotide sequence of SEQ ID NO: 18; A tenth primer set consisting of a primer having a nucleotide sequence of SEQ ID NO: 19 and a primer having a nucleotide sequence of SEQ ID NO: 20; An eleventh primer set consisting of a primer having a nucleotide sequence of SEQ ID NO: 21 and a primer having a nucleotide sequence of SEQ ID NO: 22; A twelfth primer set consisting of a primer having a nucleotide sequence of SEQ ID NO: 23 and a primer having a nucleotide sequence of SEQ ID NO: 24; A thirteenth primer set consisting of a primer having a nucleotide sequence of SEQ ID NO: 25 and a primer having a nucleotide sequence of SEQ ID NO: 26; A fourteenth primer set consisting of a primer having a nucleotide sequence of SEQ ID NO: 27 and a primer having a nucleotide sequence of SEQ ID NO: 28; A fifteenth primer set consisting of a primer having a nucleotide sequence of SEQ ID NO: 29 and a primer having a nucleotide sequence of SEQ ID NO: 30; A sixteenth primer set consisting of a primer having a nucleotide sequence of SEQ ID NO: 31 and a primer having a nucleotide sequence of SEQ ID NO: 32; A seventeenth primer set consisting of a primer having a nucleotide sequence of SEQ ID NO: 33 and a primer having a nucleotide sequence of SEQ ID NO: 34; An eighteenth primer set consisting of a primer having a nucleotide sequence of SEQ ID NO: 35 and a primer having a nucleotide sequence of SEQ ID NO: 36; A nineteenth primer set consisting of a primer having a nucleotide sequence of SEQ ID NO: 37 and a primer having a nucleotide sequence of SEQ ID NO: 38; A twentieth primer set consisting of a primer having a nucleotide sequence of SEQ ID NO: 39 and a primer having a nucleotide sequence of SEQ ID NO: 40; A twenty-first primer set consisting of a primer having a nucleotide sequence of SEQ ID NO: 41 and a primer having a nucleotide sequence of SEQ ID NO: 42; A twenty-second primer set consisting of a primer having a nucleotide sequence of SEQ ID NO: 43 and a primer having a nucleotide sequence of SEQ ID NO: 44; A twenty-third primer set consisting of a primer having a nucleotide sequence of SEQ ID NO: 45 and a primer having a nucleotide sequence of SEQ ID NO: 46; A twenty-fourth primer set consisting of a primer having a nucleotide sequence of SEQ ID NO: 47 and a primer having a nucleotide sequence of SEQ ID NO: 48; A 25 th primer set consisting of a primer having a nucleotide sequence of SEQ ID NO: 49 and a primer having a nucleotide sequence of SEQ ID NO: 50; A twenty-sixth primer set consisting of a primer having a nucleotide sequence of SEQ ID NO: 51 and a primer having a nucleotide sequence of SEQ ID NO: 52; A 27 th primer set consisting of a primer having a nucleotide sequence of SEQ ID NO: 53 and a primer having a nucleotide sequence of SEQ ID NO: 54; A twenty eighth primer set consisting of a primer having a nucleotide sequence of SEQ ID NO: 55 and a primer having a nucleotide sequence of SEQ ID NO: 56; A 29th primer set comprising a primer having a nucleotide sequence of SEQ ID NO: 57 and a primer having a nucleotide sequence of SEQ ID NO: 58; A thirtieth primer set consisting of a primer having a nucleotide sequence of SEQ ID NO: 59 and a primer having a nucleotide sequence of SEQ ID NO: 60; A thirty-first primer set consisting of a primer having a nucleotide sequence of SEQ ID NO: 61 and a primer having a nucleotide sequence of SEQ ID NO: 62; A thirty-two primer set consisting of a primer having a nucleotide sequence of SEQ ID NO: 63 and a primer having a nucleotide sequence of SEQ ID NO: 64; A thirty-third primer set consisting of a primer having a nucleotide sequence of SEQ ID NO: 65 and a primer having a nucleotide sequence of SEQ ID NO: 66; A thirty-fourth primer set consisting of a primer having a nucleotide sequence of SEQ ID NO: 67 and a primer having a nucleotide sequence of SEQ ID NO: 68; A thirty-fifth primer set consisting of a primer having a nucleotide sequence of SEQ ID NO: 69 and a primer having a nucleotide sequence of SEQ ID NO: 70; A 36th primer set consisting of a primer having a nucleotide sequence of SEQ ID NO: 71 and a primer having a nucleotide sequence of SEQ ID NO: 72; A thirty seventh primer set consisting of a primer having a nucleotide sequence of SEQ ID NO: 73 and a primer having a nucleotide sequence of SEQ ID NO: 74; A 38 th primer set consisting of a primer having a nucleotide sequence of SEQ ID NO: 75 and a primer having a nucleotide sequence of SEQ ID NO: 76; A thirty-third primer set consisting of a primer having a nucleotide sequence of SEQ ID NO: 77 and a primer having a nucleotide sequence of SEQ ID NO: 78; A 40th primer set consisting of a primer having a nucleotide sequence of SEQ ID NO: 79 and a primer having a nucleotide sequence of SEQ ID NO: 80; A forty-first primer set consisting of a primer having a nucleotide sequence of SEQ ID NO: 81 and a primer having a nucleotide sequence of SEQ ID NO: 82; A 42nd primer set consisting of a primer having a nucleotide sequence of SEQ ID NO: 83 and a primer having a nucleotide sequence of SEQ ID NO: 84; A 43rd primer set consisting of a primer having a nucleotide sequence of SEQ ID NO: 85 and a primer having a nucleotide sequence of SEQ ID NO: 86; A 44 th primer set consisting of a primer having a nucleotide sequence of SEQ ID NO: 87 and a primer having a nucleotide sequence of SEQ ID NO: 88; A forty-fifth primer set consisting of a primer having a nucleotide sequence of SEQ ID NO: 89 and a primer having a nucleotide sequence of SEQ ID NO: 90; A forty sixth primer set consisting of a primer having a nucleotide sequence of SEQ ID NO: 91 and a primer having a nucleotide sequence of SEQ ID NO: 92; A forty seventh primer set consisting of a primer having a nucleotide sequence of SEQ ID NO: 93 and a primer having a nucleotide sequence of SEQ ID NO: 94; A 48 th primer set consisting of a primer having a nucleotide sequence of SEQ ID NO: 95 and a primer having a nucleotide sequence of SEQ ID NO: 96; A 49th primer set consisting of a primer having a nucleotide sequence of SEQ ID NO: 97 and a primer having a nucleotide sequence of SEQ ID NO: 98; A fifty primer set consisting of a primer having a nucleotide sequence of SEQ ID NO: 99 and a primer having a nucleotide sequence of SEQ ID NO: 100; A 51 st primer set consisting of a primer having a nucleotide sequence of SEQ ID NO: 101 and a primer having a nucleotide sequence of SEQ ID NO: 102; A fifty-second primer set consisting of a primer having a nucleotide sequence of SEQ ID NO: 103 and a primer having a nucleotide sequence of SEQ ID NO: 104; A 53rd primer set consisting of a primer having a nucleotide sequence of SEQ ID NO: 105 and a primer having a nucleotide sequence of SEQ ID NO: 106; A 54th primer set consisting of a primer having a nucleotide sequence of SEQ ID NO: 107 and a primer having a nucleotide sequence of SEQ ID NO: 108; A fifty-fifth primer set consisting of a primer having a nucleotide sequence of SEQ ID NO: 109 and a primer having a nucleotide sequence of SEQ ID NO: 110; A sixty-first primer set consisting of a primer having a nucleotide sequence of SEQ ID NO: 111 and a primer having a nucleotide sequence of SEQ ID NO: 112; A 57th primer set consisting of a primer having a nucleotide sequence of SEQ ID NO: 113 and a primer having a nucleotide sequence of SEQ ID NO: 114; A 58th primer set consisting of a primer having a nucleotide sequence of SEQ ID NO: 115 and a primer having a nucleotide sequence of SEQ ID NO: 116; A fifty-ninth primer set consisting of a primer having a nucleotide sequence of SEQ ID NO: 117 and a primer having a nucleotide sequence of SEQ ID NO: 118; A sixty-first primer set consisting of a primer having a nucleotide sequence of SEQ ID NO: 119 and a primer having a nucleotide sequence of SEQ ID NO: 120; A sixty-first primer set consisting of a primer having a nucleotide sequence of SEQ ID NO: 121 and a primer having a nucleotide sequence of SEQ ID NO: 122; A sixty-second primer set consisting of a primer having a nucleotide sequence of SEQ ID NO: 123 and a primer having a nucleotide sequence of SEQ ID NO: 124; A sixty-third primer set consisting of a primer having a nucleotide sequence of SEQ ID NO: 125 and a primer having a nucleotide sequence of SEQ ID NO: 126; A sixty-fourth primer set consisting of a primer having a nucleotide sequence of SEQ ID NO: 127 and a primer having a nucleotide sequence of SEQ ID NO: 128; A 65th primer set consisting of a primer having a nucleotide sequence of SEQ ID NO: 129 and a primer having a nucleotide sequence of SEQ ID NO: 130; A 66th primer set consisting of a primer having a nucleotide sequence of SEQ ID NO: 131 and a primer having a nucleotide sequence of SEQ ID NO: 132; A sixty-first primer set consisting of a primer having a nucleotide sequence of SEQ ID NO: 133 and a primer having a nucleotide sequence of SEQ ID NO: 134; A sixty-first primer set consisting of a primer having a nucleotide sequence of SEQ ID NO: 135 and a primer having a nucleotide sequence of SEQ ID NO: 136; a primer having a nucleotide sequence of SEQ ID NO: 137; At least one primer set selected from the group consisting of a set or a set of primers consisting of primers having complementary sequences of respective primers, the time and cost of identification and the range of probabilities in which the significance of the results can be acknowledged In consideration of the above, Preferably, the primer set selected from the group consisting of primer sets consisting of primers having complementary sequences of the first primer set to the 69th primer set or each primer, 20 or more selected, more preferably 21 or more Selected primer set, even more wind More preferably 23 or more selected primer sets.

In an embodiment of the present invention, each of the primer sets, when amplifying the sample DNA using the primer set, as a result of analyzing the amplification product exhibited a specific level of individual specificity, when combining them Korean cattle were identified as being able to identify them. In one embodiment of the present invention, amplification of the sample DNA using 20 randomly selected primer sets and analysis of the amplification products have a probability of 1 / 736,540 (1.36 X 10 ^-6 %) of different individuals. In view of the number of Korean cattle breeding in domestic livestock farms, it was confirmed that an individual could be identified with a valid probability when using more than 20 primer sets, and the sample DNA was randomly selected using 23 primer sets. Amplification and analysis of amplification products resulted in a 1 / 5,150,922 (1.94 X 10 ^-7 %) probability that different individuals would have the same results, considering the total number of cattle raised in domestic livestock farms, 23 Using more than one primer set has been shown to be able to identify individuals with a valid probability, i.e. using more than 23 primer sets Since in the margin of error it can be reduced to within the effective range of the probability for the individual identification of beef, in the case of using the primer set is 23, while reducing the cost and time of the error range to within the effective range in terms of when the measurements were expected to be economical.

In addition, the primer set is a polymer DNA chain reaction (Polymerase Chain Reaction, PCR) using the primer set for the DNA DNA extracted from the Hanwoo, respectively, the number, size and annealing temperature of the amplified results ( T _A ), that is, since the temperature at which the double strand of DNA is separated into one strand is different, it is possible to use the 69 kinds of primer sets for selecting Korean cattle.

In addition, the primer set can identify a Hanwoo specific insertion-deletion (Indel, Insertion and Deletion) site or mismatch site that is distinguished from foreign cattle, and thus to establish a Hanwoo specific individual identification system, that is, a production history tracking system of Hanwoo Very effective.

The base sequence, the standard size of the amplification product, and the TA of the primer set corresponding to one example of the Hanwoo individual identification primer set are described in Tables 1 to 5 below.

When amplifying the sample DNA of the Korean cattle to be identified using the primer set, the probability that the number of individuals having a size other than the size of the amplified product set to the standard size is confirmed is shown in Tables 6 to 8.

In addition, in the case of randomly selecting a primer set, for example, when each of the sample DNA is amplified using the 17th and 25th primer set, the pattern of whether multiple bands of different individuals match in the result of electrophoresis of the amplification product. When the sample DNA was amplified using the primer set 45 in addition to the primer set, the probability of coinciding the pattern of multiple bands of different individuals was 12.5%. When the same method was performed using 20 primer sets, the amplification products were analyzed to have a probability of 1 / 736,540 (1.36 X 10 ^-6 %) for different individuals, and 21 primer sets. When the same method was performed using the same method, the amplification products were analyzed to have a probability of 1 / 1,416,390 (7.06 X 10 ^-7 %). When the same method was performed using two primer sets, the amplification products were analyzed to have a probability of 1 / 2,723,831 (3.67 X 10 ^-7 %) for different individuals, and 22 primer sets were used. When the same method was performed using the same method, the analysis of the amplification products revealed that the probability of different individuals having the same result was 1 / 5,150,922 (1.94 X 10 ^-7 %), As a result of considering the number of individuals raised and distributed in livestock farms, it was confirmed that when 23 or more primer sets were used, the error range could be reduced within an effective range, thereby obtaining a significant individual identification result.

In addition, the present invention provides a method for identifying a Korean cattle individual using the primer set for identifying Korean cattle individual.

In still another aspect, the present invention provides a detection kit (KIT) for identifying a Korean cattle individual comprising the primer set for identifying Korean cattle.

The identification method of the Hanwoo or Hanwoo beef to distinguish the Hanwoo or Hanwoo beef and imported cattle or imported meat is selected from the group consisting of primer set 1 to primer set 69 using one or more primer sets selected, preferably 23 or more selected primer sets Provides a way to identify each entity.

Preferably the Hanwoo individual identification method is

a) collecting a DNA sample from the cow to be identified;

b) amplifying the collected DNA sample using at least one selected primer set, preferably at least 23 selected primer sets from the group consisting of the primer set 1 to the primer set 69; And

c) analyzing the amplification products to identify individuals

Hanwoo individual identification method comprising a.

In the step a), the step of collecting the DNA sample from the cow to be identified may be performed by a method of collecting genomic DNA from the blood, semen, or juice or beef tissue of the cow to be identified. Preferably, the method may be performed by collecting genomic DNA from cow juice or beef tissue.

More specifically, the method of collecting genomic DNA may be performed by extracting DNA from blood in a case where the DNA sample is a blood sample, for example, DNA using a genomic DNA extraction kit such as a FlexiGene DNA kit. This can be done by extracting. In addition, when the DNA sample is a beef tissue, genomic DNA may be extracted from a common animal tissue. For example, the crude sample may be ground using a homogenizer or the like, and then the FlexiGene may be filtered. It can be performed by extracting DNA using genomic DNA extraction kit such as DNA kit. In addition, when the DNA sample is a semen sample, genomic DNA may be extracted from a normal animal semen. For example, after separating sperm from the animal semen, genomic DNA is extracted from the cells with respect to the sperm. It can be carried out by a method of performing a step.

B) amplifying the DNA sample using the primer set. Preferably, the DNA sample is used as a template, and the primer set for identifying the Hanwoo individual is used as a primer. Chain Reaction, PCR) may be performed by amplifying the DNA sample.

The polymerase chain reaction can be carried out under the usual reaction conditions, preferably can be carried out under the reaction conditions described in Table 8 according to the type of each primer set.

Step Temperature Reaction time First (1st) denaturation 94 ℃ 3 minutes Second (2nd) denaturation 94 ℃ 30 seconds Annealing T _A temperature of each primer set described in Tables 1-5 30 seconds Extension 72 ℃ 45 seconds Cycle go to 2nd denaturation 35 cycle Final extension 72 ℃ 5 minutes

The polymerase chain reaction may be a conventional polymerase chain reaction, and preferably, a conventional polymerase chain reaction (Conventional PCR) or a real-time polymerase chain reaction (Real-Time PCR).

The step c) may be a step of identifying an individual by analyzing an amplification product, and preferably, a method of confirming whether a plurality of DNA bands of an amplification product are plural by electrophoresis of the amplification product or the number and size of bands of DNA bands. This can be done by either checking or additionally checking the TA.

In addition, the step of identifying a Hanwoo individual in the amplification product may be performed by a method of analyzing the results obtained by electrophoresis of the amplification product, the method for determining whether there are a plurality of kinds of amplification products of the electrophoresis result. It may be a method.

For example, in the case of samples taken from Korean cattle, the size and number of DNA bands as a result of electrophoresis were confirmed based on the fact that the DNA bands resulting from the electrophoresis tend to appear as single bands or a plurality of bands. To identify the individual.

The method for checking the size of the DNA may be a conventional method for checking the size of the DNA, for example, may be performed by electrophoresis.

The detection kit for identification of a Hanwoo individual comprising the primer set for identifying an individual of Korean cattle is identified by using the primer set to amplify amplified products obtained by amplifying a DNA sample collected from a cow to be identified, and identifying the individual through the pattern of the result. It may be a kit that can be identified.

Preferably, at least one primer set selected from the group consisting of the primer set 1 to the primer set 69, preferably at least 23 selected Korean primers for identifying individual cows, and the individual specificity may be composed of detection means.

More specifically, at least one primer set selected from the group consisting of the primer set 1 to the primer set 69, preferably at least 23 selected primer sets for identifying Hanwoo individual; It may include amplification means for amplifying the DNA sample collected from the cow to be identified using the primer set and detection means for detecting the individual specificity from the amplification product amplified by the amplification means.

The DNA sample collected from the cow to be identified may be blood, semen or gravy of the cow to be identified or genomic DNA collected from a tissue of beef, preferably from a cow's juicy or beef tissue. Genomic DNA.

The identification kit may include amplification means capable of amplifying the DNA sample by using the DNA sample and the primer set, and detection means for detecting a gene expression unique to the cattle from the amplification product amplified by the amplification means. have.

The amplification means may be a conventional means for carrying out a polymerase chain reaction, and may be, for example, a reaction mixture capable of performing a polymerase chain reaction using a polymerase chain reactor. The reaction mixture may include a reaction buffer, Taq DNA polymerase, dNTP and MgCl 2, and when the polymerase chain reaction is a real-time polymerase chain reaction, additionally, fluorescence resonance energy transfer (fluorescence) as in SYBR Green I resonance energy transfer (FRET) may further comprise a means for measuring the degree of fluorescence by measuring.

In addition, the amplification means may be to include a polymerase chain reaction group in addition to the reaction mixture. The polymerase chain reactor may be a conventional polymerase chain reactor, and may be, for example, a real-time polymerase chain reactor, a thermal block polymerase chain reactor, or a micro polymerase chain reactor. However, the present invention is not limited thereto.

The detection means for detecting the specificity of the individual from the amplification product amplified by the amplification means may be a means for detecting a conventional gene marker, preferably the number of DNA bands or the DNA of the electrophoresis result of the amplification product The number, size, or the temperature at which the double helix of the amplified DNA is separated may be a means for checking the temperature.

The detection means may preferably be a device for checking the size of the amplification product, for example may be a device capable of electrophoresis of the amplification product to confirm the size of the amplification product.

Specifically, the detection means may be a detection kit for identifying individual Korean cattle, characterized in that after detecting the electrophoresis of the amplification product, the DNA band resulting from the electrophoresis is a single band or a plurality of bands. have.

As described above, in the case of using a primer set for identifying a Korean cattle individual, it is possible to identify the individual through a quick and simple method using an analytical device which is cheaper than the existing method with a significant level of probability, and thus it is cheaper than the conventional method. In addition, it is possible to identify the identity of individuals quickly even by a person who does not have expertise, so it is possible to establish a production history tracking system of Hanwoo to quickly identify and replace various risk factors and causes that can harm safe beef supply. Unlike conventional methods, unlike markers found in foreign microsatellite or SNP, unlike conventional methods, it is a Hanwoo specific primer, so it can be more accurate in case of individual identification targeting Hanwoo and foreign cows. For quick and accurate identification of the breed or individual It is expected to be widely used in various industries including livestock farming, as it has greatly helped strengthen the economic power of Hanwoo.

Hereinafter, an Example demonstrates this invention in detail.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited by the following examples.

<Examples>

Example 1 Searching for Genetic Markers Specific to Korean Cattle

Example 1-1 Preparation of BAC Library (Bacterial Arificial Chromosome library, BAC library)

Among the 26th to 35th Hanwoo Protest Groups conducted by the National Agricultural Cooperative Federation for Hanwoo, the blood of Hanwoo was judged to be excellent in meat and meat as a result of slaughter. The collected blood was refrigerated at 4 ° C. with the addition of EDTA to prevent coagulation until used for chromosomal DNA extraction.

The fabrication of the BAC library was made by previously published methods (Cai L., Taylor JF, Wing RA, Gallagher DS, Woo SS, Davis SK Construction and Characterization of a Bovine Bacterial Artificial Chromosome Library.Genomics. 29, 413-425 (1995) and Osoegawa K., Woon PY, Zhao B., Frengen E., Tateno M., Catanese JJ, de Jong PJ An improved approach for construction of bacterial artificial chromosome libraries.Genomics. 52, 1-8 (1998) ).

Chromosomal DNA of leukocyte cells was isolated and purified from the collected blood using a DNA separation kit (Genotein, South Korea) and a protocol provided by the DNA separation kit manufacturer.

After the isolated and purified chromosomal DNA is prepared with an agarose plug, an existing method using the method (An Improved Approach for Construction of Bacterial Artificial Chromosome Libraries, Genomics 52: 1-8 (1998), BAC (Bacterial Artificial Chromosome) clones containing a total of 150,000 different regions of Hanwoo chromosome were prepared with reference to the present invention).

More specifically, the isolated and purified chromosomal DNA was digested using EcoR I and Hind III restriction enzymes (NEB, USA), and the cut Hanwoo chromosome DNA was digested with pBACe3.6 vector (Accession No. U80929) and pIndigoBAC-5. Each was ligated to a vector (Epicentre, WI, USA). The ligation was carried out in 15 μl 10 X T4 ligase buffer (ligase buffer, Fermentas, Hanover, USA), 3 μl T4 ligase (ligase, Fermentas, Hanover, USA) in 50ng of pBACe3.6 vector or pIndigoBAC-5 vector. 300 ng of inserted DNA, ie, the digested Hanwoo chromosome DNA, was mixed and the total amount was adjusted to 150 μl with ultra high purity water at 16 ° C. for overnight reaction.

The recombinant vector produced by the ligation is transformed into bacterial competent cells using an electrophorator, and then plated onto LB plates. Only BAC clones containing chromosomal DNA of Hanwoo on LB plates were selected and inoculated in 96 deep well plates containing 1 ml of 2 X LB medium and incubated for 20 hours. Glycerol stock was prepared by mixing 40 μl of the culture solution and 10 μl of glycerol, and then stored in a deep freezer at -70 ° C.

Example 1-2 Isolation and Sequencing of Hanwoo BAC DNA

Isolation of BAC DNA was performed by performing both terminal sequencing on 10,272 clones of the 1500,000 clones prepared as described below.

First, the BAC clone of Hanwoo prepared above was inoculated into a 2.0 ml 96 deep well plate (Bioneer, South Korea) containing 1.5 ml of 2 X LB medium containing 12.5 μg / ml of chloramphenicol, followed by shaking. Using an incubator (shaking incubator, MegaGrow (Bioneer, South Korea), shaking culture for 18 hours at 450rpm.

The culture cultured with the shake culture was extracted using Montage BAC96 miniprep kit (Millpore) and alkaline lysis (Alkaline lysis, Birnboim , HC and Doly , JA A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic acids Res . 7 (6): 1513-1523 (1979) and Kelley, JM, Fields, CE, Craven, MB, Bocskai, D., Kim, U., Rounsley, SD and Adams, MD 1999.High throughput direct end sequencing of BAC clones. Nucleic Acids Res . 27 (6): 1539-1546.) Was used to isolate plasmid, that is, Hanwoo BAC DNA. Electrophoresis was performed on the isolated Hanwoo BAC DNA to confirm the integrity and concentration of the DNA.

After confirming the integrity and concentration of the DNA, PCR was performed using a universal sequencing primer. In more detail, the universal sequencing primer was used as a T7 primer (5'-TAATACGACTCACTATAGGG-3 ', SEQ ID NO: 139) and an engineered RP2 primer (modified RP2 primer, 5'-TACGCCAAGCTATTTAGGTGAGA-3', SEQ ID NO: 140). The PCR was performed under the conditions of Table 9 below.

Step Temperature Reaction time First (1st) denaturation 95 ℃ 1 minutes Second (2nd) denaturation 95 ℃ 30 seconds Annealing 50 ℃ 10 seconds Extension 60 ℃ 45 seconds Cycle go to 2 ^nd denaturation 35 cycle Final extension 60 ℃ 4 minutes

The DNA amplified by the PCR was subjected to capillary sequencing using an automated DNA sequencer, 3700 capillary sequencer and 3730 XL capillary sequencer (Applied Biosystems, USA), to sequence terminal sequences.

The base sequence of the BAC clone whose sequence was confirmed by the sequencing was registered in the gene bank of the National Center for Biotechnology Information (NCBI).

In order to increase the reliability of the nucleotide sequence of the BAC clone sequence identified by the sequencing, the nucleotide sequence of the vector used for constructing the clone was removed, and a clone having a phred score of 20 or more and a terminal nucleotide sequence of 100 base pairs or more was obtained. Screening was performed using cross match sofrware (http://www.phrap.org). Repeat sequence using repeat masking program (Repeat Masker-open-3-1-3, 'Wu-BLAST engine') and Rebase Current DB (Jan 20. 2006) in the selected nucleotide sequence of Hanwoo BAC clone Repeated masking, ie, removal of the repeat base sequence, was performed.

Base sequence from which the repeat base sequence was removed and Bos published in NCBI Genome data of Tarus (genome Data, AAFC01616858, AAFC01036887 , AAFC01419786, AAFC01005550, AAFC01199621, AAFC01525233, AAFC01632156, AAFC01360830, AAFC01496761, AAFC01727854, AAFC01121472, AAFC01081377, AAFC01000576, AAFC01326949, AAFC01165548, AAFC01195278, AAFC01094100, AAFC01597829, AAFC01438342, AAFC01300443, AAFC01184755, AAFC01435189, AAFC01110948, AAFC01562554, AAFC01113365, AAFC01000517, AAFC01292433, AAFC01081917, AAFC01193502, AAFC01295351, AAFC01430934, AAFC01188511, AAFC01242295, AAFC01071858, AAFC01163829, AAFC01012414, AAFC01109231, AAFC01150774, AAFC01298611, AAFC01038954, AAFC01036521, AAFC01431147, AAFC01609826, AAFC01648085, AAFC01450121, AAFC01060568, AAFC01697356, AAFC01199976, AAFC01364085, AAFC01028467, AAFC01432634, AAFC01486657, AAFC01481115, AAFC01414316, AAFC01522363, AAFC01512921, AAFC01045111, AAFC01619955, AAFC01180934, AAFC010137, AAFC01246064, AA01010744216 AA The similarity of 029) was investigated using BLAST (Basic Local Alignment Search Tool). The BLAST program used for the similarity investigation used NCBI local BLAST, and the database used NCBI Genome Bos Taurus Build 2.1.

Example  1-3: Search for Genetic Markers Unique to Korean Cattle

Hanwoo's unique genetic marker, that is, the part showing the difference between the nucleotide sequence of a cow and a foreign cow, shows the nucleotide sequence of the Hanwoo excluding the repeat base sequence and Bos published in NCBI. The genome data of Tarus were compared and searched to identify the parts representing the differences between the nucleotide sequences of Korean cattle and foreign cows, that is, an insertion-deletion (Indel, Insertion and Deletion) region and a mismatch region of 2bp or more.

Based on the comparative search result, the part showing the difference between the base sequence of Korean cattle and foreign cows using the Primer 3 program (http: //frodo.wi.mit.deu/cgi-bin/primer3/primer3_www.cgi) A sequence including a deletion (Indel, Insertion and Deletion) site or a mismatch site of 2 bp or more and a primer capable of amplifying it was determined.

< Example  2> Genetic markers unique to Hanwoo primer  making

A primer was prepared by requesting the synthesis to Bioneer (Daejeon, South Korea) based on the native marker region and the primer sequence capable of amplifying the native Hanwoo in accordance with Example 1-3.

Primers prepared by requesting the synthesis are described in Tables 10 to 14 and SEQ ID NOs: 1 to 138.

Example 3 Individual Identification of Korean Cattle

The individual identification of Hanwoo using the primers YUHW-MBL-2 (Primer Set 1) to Primer YUHW-ID-61 (Primer Set 69) prepared in Example 2 was taken from the test materials, namely, Hanwoo. Using the sample was carried out by the following method.

Example 3-1: Collection of Samples

Blood was collected from 50 Korean cattle received by a veterinarian at a slaughterhouse in Gyeongsang-do and Jeolla-do, and 10 mL of EDTA (0.5 M, pH 8.0) was added to 50 mL of collected blood to prevent coagulation of the collected blood. After being added to the tube, genomic DNA was stored at -80 ℃ until extraction.

Example 3-2 Extraction of Genomic DNA from Collected Samples

Extraction of genomic DNA from the blood sample of Example 3-1-1 was performed using a FlexiGene DNA kit (FlexiGene DNA kit, Qiagen GmbH, Hilden, Germany) and a protocol supplied by the kit manufacturer.

Example 3-3 PCR with Primer YUHW-MBL-2 to Primer YUHW-ID-61

The reaction solution for PCR reaction was genomic DNA, Taq DNA polymerase extracted from 2 μl, 3 ng of 10 X reaction buffer (reaction buffer, 100 mM Tris-Cl, 400 mL KCl, 20 mM MgCl2, pH 9.0, GenDocs, South Korea). 0.5 unit (5 unit / μl, GenDocs, South Korea), 1 μl of dNTPs mixture (2.5 mM dNTPs and 10 pmole of primer YUHW-MBL-2 (primer set 1) to primer YUHW-MBL-61 (primer set 69) After mixing, it was prepared by adding sterile distilled water to a final volume of 20 μl.

The PCR reaction solution prepared above was subjected to a PCR reaction using a MyGenie 96 Gradient thermal cycler (Bioneer, Korea) under the conditions described in Table 8 according to the primer type.

Example  3-4: Electrophoresis ( Electrophoresis With) Korean beef  discrimination

Electrophoresis was performed on the PCR product to identify the Korean beef meat using the PCR product amplified by Example 3-3.

To perform electrophoresis, a 1.2% agarose gel was prepared using 1 X TAE buffer (1 X TAE buffer, 40 mM Tris-base, 1 mM EDTA, 20 mM Acetic acid) and agarose. After loading the PCR product, electrophoresis was performed at 100 volt for 50 minutes to confirm the reaction.

After confirming the reaction by the presence or absence of the reaction, the PCR product was subjected to secondary electrophoresis using a non-denature polyacrylamide gel. The 8% non-denature polyacrylamide gel used in the secondary electrophoresis was prepared by mixing 40% acrylamide solution and 2% Bis solution (Bio-rad, USA) in a 29: 1 ratio. 4.8 ml of an amide-bis solution, 4.8 ml of sterile distilled water and 2.4 ml of 5 X TBE buffer (5 X TBE buffer, 45 mM Tris-base, 1 mM EDTA, 45 mM Boric acid) were mixed. The gel was hardened by adding 10 ul of 10% ammonium persulfate (Sigma, USA) and 10 ul of TEMED (Sigma, USA) to the prepared non-denature polyacrylamide gel.

5 μl of the PCR product of Example 3-3 was mixed with 1 μl of 6 × loading buffer, 0.25% bromophenol blue, 0.25% xylene cyanol FF, and 30% glycerol. After loading into each well of the polyacrylamide gel, electrophoresis was performed for 20 minutes at 85 volt and 1 hour 20 minutes using Mini Format Vertical Electrophoresis (Bio-rad, USA). .

The DNA size marker (DNA size marker) used to analyze the electrophoresis result was used a 100 bp DNA ladder (Bioneer, South Korea).

After completing the electrophoresis, 2 μl of EtBr (10 mg / ml) was stained and the DNA band was confirmed using ChemiImagerTM Ready (Alpha Innotech, Mississauga, Ontario, Canada), and the results are shown in FIG. 1. To FIG. 16.

The results shown in FIGS. 1 to 16 are analysis results of amplification products for individual cows that were subjected to PCR using primers YUHW-MBL-2 (primer set 1) to YUHW-ID-61 (primer set 69).

The analysis results of the PCR product for the whole Korean cattle collected in Example 3-1 are shown in Tables 15 and 16 below.

As described in Table 15 and Table 16, the amplification products for each individual cows subjected to PCR using the primers YUHW-MBL-2 (primer set 1) to YUHW-ID-61 (primer set 69) have a plurality of bands. In case of having a variety appeared in the range of 10% to 70%.

As shown in Table 17 below, the results of Table 15 and Table 16 were statistically analyzed, and the results of using a plurality of randomly selected primers were different, as expected, as the number of primer sets used increased. It was found that the probability of being identified in the subject was significantly reduced.

As shown in Table 17, when the sample DNA is amplified by using YUHW-ID-9 and YUHW-ID-17 as an example, the pattern of whether multiple bands of different individuals coincide in the result of electrophoresis of amplified products. When the sample DNA was amplified using YUHW-ID-37 in addition to the primer set, the probability of coinciding with the pattern of multiple bands of different individuals was 12.5. When the same method was performed using 21 primer sets, the amplification products were analyzed to have a 1 / 1,416,390 (7.06 X 10 ^-7 %) probability that different individuals would have the same result. When the same method was performed using the primer set, the amplification products were analyzed to have a probability of 1 / 2,723,831 (3.67 X 10 ^-7 %) for different individuals, and the same set using 22 primer sets. room When performing the, if performed by the same method, are confirmed to be the result of analyzing the amplification product of different object is ^{1 / 5,150,922 (1.94 X 10 -7} %) probability that the same result, at least 23 kinds of the primer sets In case of use, it was confirmed that individual identification is possible with a significant probability.

In addition, PCR results were performed on 50 Korean cattle using the primers YUHW-MBL-2 (primer set 1) to YUHW-ID-61 (primer set 69).

As shown in FIG. 17, as a result of analyzing an individual having a plurality of bands among the PCR products of 50 Korean cattle, an individual having a plurality of bands in the same pattern did not appear in only one individual. The pattern having the band of was different from individual to individual, and it was confirmed that the individual identification of Hanwoo was possible using the primers YUHW-MBL-2 (primer set 1) to YUHW-ID-61 (primer set 69).

Referring to the above experimental results, after preparing a primer set that can detect the insertion and deletion (Indel, Insertion and Deletion) region and mismatch region of Hanwoo, using PCR and electrophoresis using the DNA band of the electrophoresis By analyzing the differences in patterns, it is possible to compensate for the disadvantages of the expensive analysis equipment of the conventional individual identification method using microsatellite DNA markers and SNP markers, the necessity of expert personnel for equipment operation and analysis, and the inability to use them in the field quickly. In terms of reproducibility and accuracy, it can be said to be effective in identifying individual cattle.

1 is a DNA band primer YUHW-ID-1 (primer set 9) to YUHW-ID-4 (primer set 12) for DNA samples collected from 20 Hanwoo individuals according to an embodiment of the present invention The amplified product was electrophoresed to confirm the number of DNA bands.

FIG. 2 illustrates DNA bandprimers YUHW-ID-5 (primer set 13) to YUHW-ID-8 (primer set 16) of DNA samples collected from 20 Hanwoo individuals according to one embodiment of the present invention. The amplified product was electrophoresed to confirm the number of DNA bands.

Figure 3 using DNA band primers YUHW-ID-9 (primer set 17) to YUHW-ID-12 (primer set 20) for DNA samples collected from 20 Hanwoo individuals according to an embodiment of the present invention The amplified product was electrophoresed to confirm the number of DNA bands.

Figure 4 using DNA band primers YUHW-ID-13 (primer set 21) to YUHW-ID-16 (primer set 24) for DNA samples collected from 20 Hanwoo individuals according to an embodiment of the present invention The amplified product was electrophoresed to confirm the number of DNA bands.

FIG. 5 illustrates DNA bandprimers YUHW-ID-17 (primer set 25) to YUHW-ID-20 (primer set 28) for DNA samples collected from 20 Hanwoo individuals according to one embodiment of the present invention. The amplified product was electrophoresed to confirm the number of DNA bands.

Figure 6 using DNA band primers YUHW-ID-21 (primer set 29) to YUHW-ID-24 (primer set 32) for DNA samples collected from 20 Hanwoo individuals according to an embodiment of the present invention The amplified product was electrophoresed to confirm the number of DNA bands.

FIG. 7 illustrates DNA band primers YUHW-ID-25 (primer set 33) to YUHW-ID-27 (primer set 35) of DNA samples collected from 20 Korean cattle, according to an embodiment of the present invention. The amplified product was electrophoresed to confirm the number of DNA bands.

FIG. 8 illustrates DNA bandprimers YUHW-ID-28 (primer set 36) to YUHW-ID-31 (primer set 39) of DNA samples collected from 20 Hanwoo individuals according to one embodiment of the present invention. The amplified product was electrophoresed to confirm the number of DNA bands.

FIG. 9 is a DNA band primer YUHW-ID-32 (primer set 40) to YUHW-ID-35 (primer set 43) for DNA samples collected from 20 Hanwoo individuals according to one embodiment of the present invention. The amplified product was electrophoresed to confirm the number of DNA bands.

FIG. 10 illustrates DNA bandprimers YUHW-ID-36 (primer set 44) to YUHW-ID-39 (primer set 47) of DNA samples collected from 20 Hanwoo individuals according to one embodiment of the present invention. The amplified product was electrophoresed to confirm the number of DNA bands.

FIG. 11 illustrates DNA bandprimers YUHW-ID-40 (primer set 48) to YUHW-ID-43 (primer set 51) of DNA samples collected from 20 Hanwoo individuals according to one embodiment of the present invention. The amplified product was electrophoresed to confirm the number of DNA bands.

FIG. 12 illustrates DNA bandprimers YUHW-ID-44 (primer set 52) to YUHW-ID-47 (primer set 55) of DNA samples collected from 20 Hanwoo individuals according to one embodiment of the present invention. The amplified product was electrophoresed to confirm the number of DNA bands.

FIG. 13 illustrates DNA bandprimers YUHW-ID-48 (primer set 56) to YUHW-ID-51 (primer set 59) of DNA samples collected from 20 Hanwoo individuals according to one embodiment of the present invention. The amplified product was electrophoresed to confirm the number of DNA bands.

FIG. 14 illustrates the use of DNA band primers YUHW-ID-52 (primer set 60) to YUHW-ID-55 (primer set 63) for DNA samples collected from 20 Hanwoo individuals according to one embodiment of the present invention. The amplified product was electrophoresed to confirm the number of DNA bands.

FIG. 15 is a diagram illustrating DNA samples collected from 20 Hanwoo individuals using DNA band primers YUHW-ID-56 (primer set 64) to YUHW-ID-59 (primer set 67) according to one embodiment of the present invention. The amplified product was electrophoresed to confirm the number of DNA bands.

FIG. 16 is a DNA band primer YUHW-ID-60 (primer set 68) and YUHW-ID-61 (primer set 69) for DNA samples collected from 20 Hanwoo individuals according to an embodiment of the present invention. The amplified product was electrophoresed to confirm the number of DNA bands.

17 is a result of confirming whether a plurality of DNA bands are obtained by electrophoresis of an amplification product amplified using primer sets 1 to 69 against DNA samples collected from 50 Korean cattle, according to an embodiment of the present invention. , 69 is a set of 69 primer, the length is 50 Hanwoo, colored dots indicate that the DNA band of the amplification product is plural.

<110> Industry-Academic Cooperation Yeungnam University <120> A NEW PRIMER SET AND A METHOD OF SELECTING AN HANWOO INDIVIDUAL          BY USING THE SAID PRIMER SET <130> DPP20073369KR <160> 140 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-MBL-2-F-Primer <400> 1 tgatcaaggt cacaggaagg 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-MBL-2-R-Primer <400> 2 cgagaaacta gggtcgaagt 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-MBL-3-F-Primer <400> 3 ctctgaggtg agggaacagg 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-MBL-R-Primer <400> 4 acgagacgaa ttgtggtacg 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-MBL-5-F-Primer <400> 5 cccaagctcc aaaagacagt 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-MBL-5-R-Primer <400> 6 tgtcccctta cagtcgtgaa 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-MBL-6-F-Primer <400> 7 caagcttggg ctttcctaaa 20 <210> 8 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> YUHW-MBL-6-R-Primer <400> 8 tctcctctct acccctact 19 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-MBL-7-F-Primer <400> 9 cagccaggaa tcaggttttc 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-MBL-7-R-Primer <400> 10 tttcctgtag aacacagggc 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-MBL-8-F-Primer <400> 11 ttcccagtct caaggcactc 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-MBL-8-R-Primer <400> 12 ttcatgtgac ccggtaaacg 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-MBL-9-F-Primer <400> 13 tcctttttgg catcttggag 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-MBL-9-R-Primer <400> 14 cgacaaagga caaacgacct 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-MBL-10-F-Primer <400> 15 acagcacttg gcttcctcat 20 <210> 16 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> YUHW-MBL-10-R-Primer <400> 16 attaacaggt gtctcacgac tc 22 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-1-F-Primer <400> 17 ccatcccata ggcagtcagt 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-1-R-Primer <400> 18 tccaaagttc cgggacgata 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-2-F-Primer <400> 19 ccctgctcca gtgacttctt 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-2-R-Primer <400> 20 taggaaaaga cgaggaaggg 20 <210> 21 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-3-F-Primer <400> 21 gcggctataa ttaacactag caaa 24 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-3-R-Primer <400> 22 tattagacca ccgtcaaccg 20 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-4-F-Primer <400> 23 ttgggcaaaa tgttacatgc 20 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-4-R-Primer <400> 24 ggtactcttt ttcccctgtc 20 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-5-F-Primer <400> 25 tgcttctctc ctgatgatgg 20 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-5-R-Primer <400> 26 aaccttcaaa ccccgtattt 20 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-6-F-Primer <400> 27 agcgtgctga aaggtgatct 20 <210> 28 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-6-R-Primer <400> 28 aaggaagttc ccggtaatac 20 <210> 29 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-7-F-Primer <400> 29 agtggcattt gacacaatcg 20 <210> 30 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-7-R-Primer <400> 30 gagttgtaaa agtaaactta ccaa 24 <210> 31 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-8-F-Primer <400> 31 cttgcagcct ctaagcaagg 20 <210> 32 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-8-R-Primer <400> 32 gtccttacct tgggtacaga 20 <210> 33 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-9-F-Primer <400> 33 tccacgtttt tctccaccat 20 <210> 34 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-9-R-Primer <400> 34 aagatggaaa ccaaaccgct 20 <210> 35 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-10-F-Primer <400> 35 aaaatcctaa acttgccaaa tga 23 <210> 36 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-10-R-Primer <400> 36 aggttacgtt ttcattgtca act 23 <210> 37 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-11-F-Primer <400> 37 cggcagtaac cacaggtagg 20 <210> 38 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-11-R-Primer <400> 38 ctttcgtgta acgttcctgg 20 <210> 39 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-12-F-Primer <400> 39 tggtcaccct tccttgtgat 20 <210> 40 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-12-R-Primer <400> 40 tcaccccaga cctcagtgtg 20 <210> 41 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-13-F-Primer <400> 41 tcagacccac cgtataaaga atg 23 <210> 42 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-13-R-primer <400> 42 ctcttagaaa cagttaacga aact 24 <210> 43 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-14-F-Primer <400> 43 ccacctactt cagtgccaat 20 <210> 44 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-14-R-Primer <400> 44 ggaagagaat ccttggtacg t 21 <210> 45 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-15-F-Primer <400> 45 ctttggtgag aggagggtga 20 <210> 46 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-15-R-Primer <400> 46 acacggtacc catagtagta 20 <210> 47 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-16-F-Primer <400> 47 agttccgcca tgtccttatg 20 <210> 48 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-16-R-Primer <400> 48 accactaagt ttcgaacacg 20 <210> 49 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-17-F-Primer <400> 49 ggaaactgtt ccaggcagaa 20 <210> 50 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-17-R-Primer <400> 50 ggtaaagtgg acagacgtct 20 <210> 51 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-18-F-Primer <400> 51 gattttcttc tccaccattg c 21 <210> 52 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-18-R-Primer <400> 52 agtgacggta caatcggcat 20 <210> 53 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-19-F-Primer <400> 53 tctgggctaa actcaacctc a 21 <210> 54 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-19-R-Primer <400> 54 acacagttgg tctcagtaga ct 22 <210> 55 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-20-F-Primer <400> 55 ttttgtgggc cttaagttgg 20 <210> 56 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-20-R-Primer <400> 56 gcaaacagtg tcccttccta 20 <210> 57 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-21-F-Primer <400> 57 tacagggtgc cagttgtctg 20 <210> 58 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-21-R-Primer <400> 58 gaaagaagtg tgacacgggt 20 <210> 59 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-22-F-Primer <400> 59 ttgcctcctc ctatcaagtc a 21 <210> 60 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-22-R-Primer <400> 60 cttacgaacc attagtgacc c 21 <210> 61 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-23-F-Primer <400> 61 caaatgtagc caaacggtga 20 <210> 62 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-23-R-Primer <400> 62 cacgaggtgt acgtctttct 20 <210> 63 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-24-F-Primer <400> 63 gccatttcac ctggacctaa 20 <210> 64 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-24-R-Primer <400> 64 acaccgacaa tgaaacggag a 21 <210> 65 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-25-F-Primer <400> 65 gcctgacttg agaaggatgc 20 <210> 66 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-25-R-Primer <400> 66 actgttcgac ttgagggagg 20 <210> 67 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-26-F-Primer <400> 67 ctgcttcccc ttgaagtctg 20 <210> 68 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-26-R-Primer <400> 68 ttgtttgggg ttcttcgtct 20 <210> 69 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-27-F-Primer <400> 69 gagggaggac atggtctcag 20 <210> 70 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-27-R-Primer <400> 70 tttatccctc cccattccgg 20 <210> 71 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-28-F-Primer <400> 71 agtttccctt ggctttggat 20 <210> 72 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-28-R-Primer <400> 72 tttctcacgg tccgaatcgt 20 <210> 73 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-29-F-Primer <400> 73 aacccgtgac tataatggtg gt 22 <210> 74 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-29-R-Primer <400> 74 ggtttcttca gtcctcgacc 20 <210> 75 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-30-F-Primer <400> 75 cctaggatcg aggcacacat 20 <210> 76 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-30-R-Primer <400> 76 tctttagggt cctccagcta 20 <210> 77 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-31-F-Primer <400> 77 tcgttgcttg gctttgataa 20 <210> 78 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-31-R-Primer <400> 78 caaggagaac acttacgcca 20 <210> 79 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-32-F-Primer <400> 79 tcctctgtag gcatccatcc 20 <210> 80 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-32-R-Primer <400> 80 gtcgaattac cggtacaagt 20 <210> 81 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-33-F-Primer <400> 81 ctgtggttgg cagtgtctgt 20 <210> 82 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-33-R-Primer <400> 82 aaagtggttg ggtaaatccg 20 <210> 83 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-34-F-Primer <400> 83 gccaggcaac atttatgtga 20 <210> 84 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-34-R-Primer <400> 84 ccgattcgct ttttagtggt 20 <210> 85 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-35-F-Primer <400> 85 agaagctgca gttcctggac 20 <210> 86 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-35-R-Primer <400> 86 gacgtacagg aagaaggagg 20 <210> 87 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-36-F-Primer <400> 87 gagctgaaaa caggcagacc 20 <210> 88 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-36-R-Primer <400> 88 ctattggagg agacgggtgt 20 <210> 89 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-37-F-Primer <400> 89 tgatgaacgt aggcaccaaa 20 <210> 90 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-37-R-Primer <400> 90 ggtctccggt acgataaaga 20 <210> 91 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-38-F-Primer <400> 91 atctggaagg gctcaacaga 20 <210> 92 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-38-R-Primer <400> 92 agagtgctta ttactccggg 20 <210> 93 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-39-F-Primer <400> 93 tttttcaagg acctgcatcc 20 <210> 94 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-39-R-Primer <400> 94 tcagtcctct actgggaggt 20 <210> 95 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-40-F-Primer <400> 95 agcactggtt catcccattc 20 <210> 96 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-40-R-Primer <400> 96 aagaccgtga atcgagacgt 20 <210> 97 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-41-F-Primer <400> 97 ttccagaggg aatggaagtg 20 <210> 98 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-41-R-Primer <400> 98 gtcactcaaa acggtcgtac 20 <210> 99 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-42-F-Primer <400> 99 gtcttctggt ggtgggagaa 20 <210> 100 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-42-R-Primer <400> 100 ttttgaccct gaacacgtcc 20 <210> 101 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-43-F-Primer <400> 101 aggtcccttt ccaggagcta 20 <210> 102 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-43-R-Primer <400> 102 aagacggttt gtccaatggg 20 <210> 103 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-44-F-Primer <400> 103 aggcgcttag aaccacaaaa 20 <210> 104 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-44-R-Primer <400> 104 tcacaaccct gacctgttgg 20 <210> 105 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-45-F-Primer <400> 105 tctcccaacc caccctaaat 20 <210> 106 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-45-R-Primer <400> 106 cgacttgaaa cggatgtccg 20 <210> 107 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-46-F-Primer <400> 107 acatcatttt tgcccagagc 20 <210> 108 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-46-R-Primer <400> 108 ggttccgtcc gtgatcaaaa 20 <210> 109 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-47-F-Primer <400> 109 tccaggatca atgctttcaa 20 <210> 110 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-47-R-Primer <400> 110 aaactcggac ctagaacgaa 20 <210> 111 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-48-F-Primer <400> 111 acaagtttgg ggaaccagtg 20 <210> 112 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-48-R-Primer <400> 112 ctcacctagt tcgggtagta 20 <210> 113 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-49-F-Primer <400> 113 ctggtgaaca gacgaggtga 20 <210> 114 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-49-R-Primer <400> 114 aaaacagact tcctggaccc 20 <210> 115 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-50-F-Primer <400> 115 ttttgagctg tctttgaagt attga 25 <210> 116 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-50-R-Primer <400> 116 cgtatcatcc tttgaccgga c 21 <210> 117 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-51-F-Primer <400> 117 cactttgctc cctcattgat 20 <210> 118 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-51-R-Primer <400> 118 aacaagactt ggacgtcgaa 20 <210> 119 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-52-F-Primer <400> 119 ttgatgaaat tccctccttg 20 <210> 120 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-52-R-Primer <400> 120 acgatggaat tacgactctt ctt 23 <210> 121 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-53-F-Primer <400> 121 tggtgcccag ttaatgtcag 20 <210> 122 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-53-R-Primer <400> 122 ctcctctttt cgcttccact 20 <210> 123 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-54-F-Primer <400> 123 ggcactgatg ccttgttctt 20 <210> 124 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-54-R-Primer <400> 124 aacttttcac caactttgtc tga 23 <210> 125 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-55-F-Primer <400> 125 accaccgtac cctgaatcac 20 <210> 126 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-55-R-Primer <400> 126 cttggtagac aactcggcaa 20 <210> 127 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-56-F-Primer <400> 127 tgggctcaga tcctacttgg 20 <210> 128 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-56-R-Primer <400> 128 acaaacgagg acagagaggg 20 <210> 129 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-57-F-Primer <400> 129 gcacttgcct tctcatctcc 20 <210> 130 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-57-R-Primer <400> 130 attccgtcta agggtcggat 20 <210> 131 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-58-F-Primer <400> 131 ttttgcattt cgcttaacca 20 <210> 132 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-58-R-Primer <400> 132 accttaccag gaactagaca c 21 <210> 133 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-59-F-Primer <133> 133 ttgctttccg tgaaacttcc 20 <210> 134 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-59-R-Primer <400> 134 cactatcggt tcccttgagt aa 22 <210> 135 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-60-F-Primer <400> 135 ggatctgtta ctgggaagac c 21 <210> 136 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-60-R-Primer <400> 136 ggtagttcct taaatttgtc gt 22 <210> 137 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-61-F-Primer <400> 137 tgaattgcat ctgcctcatt 20 <210> 138 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> YUHW-ID-61-R-Primer <400> 138 taagacttta ggtaggggtg a 21 <139> <211> 20 <212> DNA <213> Artificial Sequence <220> <223> T7 primer <400> 139 taatacgact cactataggg 20 <210> 140 <211> 23 <212> DNA <213> Artificial Sequence <220> 223 RP2 primer <400> 140 tacgccaagc tatttaggtg aga 23  

Claims (6)

A primer set for identifying Hanwoo individual, which is a set of 23 or more primers selected from the group consisting of the first to 69th primer sets of Tables 1a to 1e. TABLE 1a

TABLE 1b

TABLE 1c

TABLE 1d

TABLE 1e

delete a) collecting a DNA sample from the cow to be identified; b) amplifying the collected DNA sample using at least 23 primer sets selected from the group consisting of primer set 1 to primer set 69 according to claim 1; And c) analyzing the amplification products to identify individuals Hanwoo individual identification method comprising a. The method of claim 3, Wherein c) step is performed by electrophoresis the amplification product to determine whether the DNA band is plural Hanwoo individual identification method. Primer set for identifying a Hanwoo individual 23 or more selected from the group consisting of primer set 1 to primer set 69 according to claim 1; A detection kit for identifying individual Korean cattle, comprising amplification means for amplifying DNA samples collected from cows to be identified using the primer set, and detection means for detecting Hanwoo individual specificity from a product amplified by the amplification means. . The method of claim 5, The detection means is a detection kit for identifying individual Korean cattle, characterized in that after the electrophoresis of the amplification product, means for confirming whether the DNA band resulting from the electrophoresis is a single band or a plurality of bands.

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