KR100415080B1 - Primer related marbling score of Hanwoo and test process of marbling score using thereof - Google Patents

Primer related marbling score of Hanwoo and test process of marbling score using thereof Download PDF

Info

Publication number
KR100415080B1
KR100415080B1 KR10-2000-0065925A KR20000065925A KR100415080B1 KR 100415080 B1 KR100415080 B1 KR 100415080B1 KR 20000065925 A KR20000065925 A KR 20000065925A KR 100415080 B1 KR100415080 B1 KR 100415080B1
Authority
KR
South Korea
Prior art keywords
dna
hanwoo
primer
primers
fat
Prior art date
Application number
KR10-2000-0065925A
Other languages
Korean (ko)
Other versions
KR20020035678A (en
Inventor
여정수
Original Assignee
학교법인 영남학원
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 학교법인 영남학원 filed Critical 학교법인 영남학원
Priority to KR10-2000-0065925A priority Critical patent/KR100415080B1/en
Publication of KR20020035678A publication Critical patent/KR20020035678A/en
Application granted granted Critical
Publication of KR100415080B1 publication Critical patent/KR100415080B1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/124—Animal traits, i.e. production traits, including athletic performance or the like
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

본 발명은 한우의 근내지방도 연관 프라이머 및 그를 이용한 한우 근내지방도 성적 검사방법에 관한 것으로, AFLP에 사용될 프라이머와 어댑터를 합성한 후 그 프라이머를 제한 효소로 절단한 한우의 genomic DNA와 반응시켜 PCR을 수행함으로써 근내지방도 성적이 높은 집단과 낮은 집단에서 특이적으로 차별화되는 특이적 DNA 표지인자를 획득하고 염기서열을 분석한 후 그를 이용하여 제조한 15mer의 근내지방도 연관 프라이머를 이용하여 한우의 육질을 검사한 결과, 보다 손쉽고 경제적이며 정확한 한우 육질 검사방법, 특히 근내지방도 성적을 검사하는 방법을 제공하는 뛰어난 효과가 있다.The present invention relates to a Korean native beef myofascial fat associated primers and a method for testing Korean bovine myocardial fat using the same. After the synthesis of primers and adapters for AFLP, the primers are reacted with genomic DNA of Hanwoo digested with restriction enzymes to perform PCR. By obtaining specific DNA markers that are differentiated specifically from high and low myocardial fat groups and analyzing the sequencing, the meat quality of Hanwoo was examined using 15mer intramuscular fat related primers. As a result, there is an excellent effect of providing a easier, more economical and accurate method for testing the quality of beef cattle, especially the intramuscular fat score.

Description

한우의 근내지방도 연관 프라이머 및 그를 이용한 한우 근내지방도 성적 검사방법 {Primer related marbling score of Hanwoo and test process of marbling score using thereof}Primer related marbling score of Hanwoo and test process of marbling score using

본 발명은 한우의 근내지방도 연관 프라이머 및 그를 이용한 한우 근내지방도 성적 검사방법에 관한 것이다. 더욱 상세하게는, 본 발명은 한우의 혈액으로부터 genomic DNA를 추출하고 그로부터 근내지방도 관련 특이적 DNA 마커를 추출한 후 그 염기서열을 분석하여 육질을 결정하는 중요 요인인 근내지방도 연관 프라이머를 개발하고 그를 이용하여 보다 바람직하고 손쉽게 한우 육질, 특히 근내지방도를 검사하는 방법에 관한 것이다.The present invention relates to an intramuscular fat degree associated primer of Hanwoo and a method for testing the intramuscular fatness of Hanwoo using the same. More specifically, the present invention extracts genomic DNA from the blood of Hanwoo, extracts specific DNA markers related to intramuscular adipose from thereafter, and analyzes the sequencing to develop a primer related to intramuscular fat, which is an important factor for determining meat quality. More preferably and easily relates to a method of inspecting the quality of beef cattle, especially intramuscular fat.

한우 (Hanwoo)는 한국 고유의 소품종으로서 수천년간 한국 풍토에 적응하여 그에 적합한 체질로 고정되어 온 주요 가축이다. 한국에 수입되는 외국산 육류 물량은 수입자유화 이후로 꾸준히 증가하여 국내 축산업을 위협하고 있는 실정이다. 이러한 상황을 타개하기 위해서는 한우 육질의 개선과 일등품 우육의 선별적 사육이 필수적이다.Hanwoo (Hanwoo) is a unique prop species of Korea, which has been adapted to the Korean climate for thousands of years and has been fixed in the proper constitution. The amount of foreign meat imported into Korea has increased steadily since the import liberalization, threatening the domestic livestock industry. In order to overcome this situation, it is necessary to improve the quality of beef cattle and selectively raise first-class beef cattle.

일반적으로 쇠고기의 육질을 결정하는 요인으로는 근내지방침착도 (marbling score), 육질의 색과 밝기, 육질의 보습성 및 지방의 조성 등이 있는데, 이중에서도 근내지방도는 육질을 결정하는 가장 중요한 요인이다. 육질을 확인하기 위하여 종래에는 소를 도살하여야 확인할 수 있었으나 매우 비효율적인 것으로, 그에 따라 우수한 품질등급의 비육우를 사육하기 위한 한우 육질 판별 방법이 요구되어 왔다. 유전공학의 발달에 따른 DNA 차원의 실험기법들의 발전이 지속적으로 이루어져 왔으며, 이러한 실험 기법들의 발전을 토대로 가축의 유전적 특성이나 능력개량을 위한 다양한 DNA typing 기술들이 개발되어 축산에 이용되고 있다. 이러한 유전공학적 기법에는 PCR (Polymerase Chain Reaction)을 이용한 RAPD (Random Amplified Polymorphic DNA), AP-PCR (Arbitratily Primer-Polymerase Chain Reaction) 및 AFLP (Amplified Fragment Length Polymorphism)등이 있고, PCR을 이용하지 않는 방법의 대표적인 것으로는 DNA fingerprinting (DNA typing)과 RFLP (Restriction Fragment Length Polymorphism)이 있다. PCR은 간편한 처리 방법 및 짧은 시간에 많은 수의 샘플 결과를 확인 할 수 있다는 장점이 있으나, 재현성에서 문제점을 나타내고 있고, 제한 효소로 처리하여 생긴 DNA 단편들을 막에 전사한 후 표식된 프로브를 사용하여 DNA 다형성을 검출하는 RFLP에 의한 fingerprinting은 PCR에 기초를 두고 있지 않기 때문에 DNA typing용으로는 별다른 불편한 점이 없으나, 유전자지문에 비해 여러 가지 프로브에 대한 적용이 어려운 이유로 다양한 다형성 (polymorphism; 생물 집단내의 유전적 변이가 개체간에도 구별될 정도로 다양하다는 것)을 관찰할 수가 없어, DNA 마커의 cloning과 mapping에는 취약점을 가지고 있다. 최근 Vos등 (1995)이 개발한 DNA 지문 방법인 AFLP 기술은 개념상으로 실험방법이 까다로운 Southern blotting에 의존하는 힘든 기술인 RFLP와 reaction condition에 아주 민감하고 재현성이 떨어지는 RAPD 기술의 장점만을 조합시킨 분자생물학적 기술로서, Jeffreys등 (1985)이 발표한 DNA fingerprinting의 기술보다 손쉬운 실험과정으로 다양한 DNA polymorphism을 얻을 수 있어, 새로운 가축의 DNA 연구에 중요한 방법으로 인식되고 있으며, 최근 여러 가축에서 능력개량을 위한 최신의 기법으로 적용되고 있다. 또한, DNA polymorphism에 의해 나타난 DNA 마커는 DNA fingerprinting에서는 DNA typing용으로 사용하는데 별다른 불편함이 없으나DNA 마커 자체의 클로닝을 통한 DNA 서열을 밝히는데는 많은 어려움이 있다. 그러나, AFLP 실험을 통해 나타난 DNA 마커는 겔 상에서 잘라내어 바로 클로닝하여 DNA 구조의 시퀀싱이 가능하므로 활용범위가 넓은 것으로 알려져 있다.In general, the determinants of beef quality include marbling score, color and brightness of meat, moisture retention and fat composition. Among these, muscle fatness is the most important factor that determines meat quality. to be. In order to check the meat quality in the past, butchering the cows could be confirmed, but it is very inefficient. Accordingly, there has been a demand for a method for determining the quality of beef cattle for raising beef cattle of excellent quality grade. The development of DNA-level experimental techniques has been continuously made in accordance with the development of genetic engineering. Based on the development of these experimental techniques, various DNA typing techniques have been developed and used for animal husbandry to improve the genetic characteristics and ability of livestock. Such genetic engineering techniques include random amplified polymorphic DNA (RAPD) using PCR (Polymerase Chain Reaction), Arbitratily Primer-Polymerase Chain Reaction (AP-PCR), and Amplified Fragment Length Polymorphism (AFLP). Representatives of DNA include DNA fingerprinting (DNA typing) and Restriction Fragment Length Polymorphism (RFLP). PCR has the advantage of easy processing method and a large number of sample results in a short time, but shows a problem in reproducibility, using a labeled probe after transferring the DNA fragments generated by the restriction enzyme to the membrane Fingerprinting by RFLP, which detects DNA polymorphism, is not based on PCR, so it is not inconvenient for DNA typing.However, it is difficult to apply various probes to genetic fingerprinting. Redundant mutations can vary from one individual to another, making them vulnerable to cloning and mapping of DNA markers. AFLP, a DNA fingerprinting method developed recently by Vos et al. (1995), combines the advantages of RFLP, a tough technique that relies on Southern blotting, which is conceptually challenging, and RAPD technology, which is very sensitive to reaction conditions and is less reproducible. As a technique, various DNA polymorphisms can be obtained through an easier experimental process than the technique of DNA fingerprinting published by Jeffreys et al. (1985), and it is recognized as an important method for researching new livestock DNA. It is applied by technique. In addition, DNA markers exhibited by DNA polymorphism are not inconvenient to use for DNA typing in DNA fingerprinting, but there are many difficulties in identifying DNA sequences through cloning of DNA markers themselves. However, DNA markers shown through AFLP experiments are known to be widely used because they can be cut on a gel and immediately cloned to sequence DNA structures.

DNA 다형성 (DNA polymorphism)은 단백질 변이의 경우와는 달리 가축의 발육시기 및 발육단계에 영향을 받지 않으며 게놈 중 대부분의 DNA는 형질 (단백질, 효소 등)로서 발현되지 않고 있고 형질로 발현되지 않는 부분이라도 DNA 수준에서는 분석이 가능하므로 DNA를 이용하면 유리하고 정확하게 분석할 수 있다. 특히, DNA 변이를 검출하는데 주로 사용되는 프로브는 개체간의 차이도 검출할 수 있을 정도로 고감도이다. 본 발명에서는 AFLP를 통하여 다수의 경제형질 연관 DNA 마커를 규명하고, 이들 DNA 마커에 대한 염기서열 분석을 완료하여 일부 프라이머의 개발을 수행하였다.Unlike protein mutations, DNA polymorphism is not affected by the timing and stage of development of livestock, and most DNA in the genome is not expressed as a trait (protein, enzyme, etc.) and is not expressed as a trait. Even DNA can be analyzed at the DNA level, so DNA can be analyzed advantageously and accurately. In particular, probes mainly used to detect DNA mutations are highly sensitive enough to detect differences between individuals. In the present invention, a plurality of economic trait DNA markers were identified through AFLP, and sequencing of these DNA markers was completed to develop some primers.

현재 우리나라에서 한우의 육질을 판단하는 기준 등급은 근내지방도에 따라 1+등급, 1등급, 2등급 및 3등급으로 나누어지는데 본 발명에서는 등급이 높은 (1+ 또는 1등급) 개체와 등급이 낮은 (3등급) 개체들에서 상기 PCR 방법 중 AFLP DNA 분석 기술을 이용하여 유효한 차이가 있는 DNA 마커를 찾아낸 후 이 DNA 마커의 염기서열을 분석하여 이 염기서열내에서 근내지방도에 연관된 표식을 쉽게 확인할 수 있는 프라이머를 개발하였다. 이 발명은 DNA (유전자)는 소의 발육 단계에 따라서 불변하는 특성을 이용한 것으로 어린 한우 송아지 개체를 본 발명 프라이머를 사용하여 판별하고 선택적으로 사육함으로써 실질적으로 농가의 소득을 향상시키고자 수행된 것이다. 특히, 개발되어진 본 발명 프라이머는 외국 축우와의 경쟁에서 우위를 확보하는데 있어 필수조건인 우리나라 고유품종 한우의 경제형질 연관 DNA 마커에 대한 개발로써 그 의미가 있다.At present, the standard grade for determining the quality of Korean beef is divided into 1+ grade, 1 grade, 2 grade and 3 grade according to intramuscular fat. In the present invention, high grade (1+ or 1 grade) individuals and low grade ( Level 3) individuals can find the DNA markers with valid differences among the PCR methods using AFLP DNA analysis technology, and then analyze the nucleotide sequences of these DNA markers to easily identify markers related to intramuscular fat within these nucleotide sequences. Primers were developed. This invention is to use DNA (gene) is invariant characteristics according to the development stage of the cow was carried out to substantially improve the income of farmers by discriminating and selectively breeding young Hanwoo calf individuals using the primer of the present invention. In particular, the developed primer of the present invention has a meaning as a development of the DNA marker associated with the economic traits of Korean native varieties of Korean native cattle, which is an essential condition to secure an advantage in competition with foreign cattle.

따라서, 본 발명의 목적은 한우 genomic DNA로부터 추출한 근내지방도 연관 특이적 DNA 마커의 염기서열을 제공하는데 있다. 본 발명의 다른 목적은 한우 품종의 육질을 판별할 수 있는 근내지방도 연관 프라이머 및 그 염기서열을 제공하는데 있다. 본 발명의 또다른 목적은 상기 본 발명 프라이머를 사용하여 한우의 근내지방도 성적을 검사하는 방법을 제공하는데 있다.Accordingly, an object of the present invention is to provide a nucleotide sequence of a specific DNA marker associated with intramuscular fat extracted from Hanwoo genomic DNA. Another object of the present invention is to provide a primer and its base sequence associated with intramuscular fat that can determine the meat quality of Hanwoo cultivar. Another object of the present invention to provide a method for testing the intramuscular fat score of Hanwoo using the primer of the present invention.

본 발명의 상기 목적은 한우의 백혈구로부터 genomic DNA를 분리하고TaqⅠ 및EcoRⅠ제한 효소를 첨가하여 DNA 이중 침지를 실시한 후 그 DNA 말단에 이중가닥 어댑터를 라이게이션시켜 한우 DNA를 준비하고, AFLP에 사용할 프라이머를 합성한 후 상기 DNA 시약과 반응시켜 AFLP-PCR을 수행하고 그 중 근내지방도 성적이 높은 집단과 낮은 집단에서 특이적 반응을 보이는 DNA 마커를 분리하고 그 염기서열을 분석한 후 그로부터 유래한 한우 근내지방도 연관 프라이머를 개발하고 그를 이용한 한우 근내지방도 성적 검사방법을 제공함으로써 달성되었다.The above object of the present invention is to isolate the genomic DNA from the leukocytes of Hanwoo, and add double Taq I and Eco Rl restriction enzyme to perform DNA double immersion and ligated a double-stranded adapter at the end of the DNA to prepare Hanwoo DNA, AFLP After synthesizing the primers to be used, AFLP-PCR was performed by reacting with the DNA reagent. Among them, DNA markers showing specific reactions in the high and low myocardial fat scores were isolated and the sequencing was analyzed. Hanwoo intramuscular fat was also achieved by developing an associated primer and providing a method for the evaluation of intramuscular fat using Hanwoo.

이하, 본 발명의 구성을 상세히 설명한다.Hereinafter, the configuration of the present invention will be described in detail.

도 1은 AFLP의EcoRⅠ 9/TaqⅠ 2 프라이머를 이용하여 한우 [Hanwoo (Korea cattle)]의 근내지방도 성적이 높은 집단과 낮은 집단에서 차별화되는 특이적 DNA 마커를 PCR로 확인한 결과이다.1 shows AFLPEcoRⅠ 9 /TaqI PCR results showed specific DNA markers differentiated between high and low populations of Hanwoo (Korea cattle) using 2 primers.

도 2는 AFLP의EcoRⅠ9/TaqⅠ2 프라이머를 이용하여 PCR을 수행한 결과 근내지방도 성적이 높은 집단과 낮은 집단에서 차별화되는 근내지방도 연관 139bp DNA 특이적 마커의 염기서열을 나타낸다.Figure 2 shows the nucleotide sequence of the 139 bp DNA specific marker associated with intramuscular fat, which is differentiated from the high and low myopia fat score groups by PCR using the Eco RI9 / Taq I2 primer of AFLP.

도 3은EcoRⅠ9/TaqⅠ2 프라이머로 규명된 상기 도 2의 근내지방도 연관 139bp DNA 마커의 염기서열을 분석하여 제작한 프라이머를 나타낸다.Figure 3 shows a primer prepared by analyzing the nucleotide sequence of the 139 bp DNA marker associated with the intramuscular fat diagram of Figure 2 identified as Eco RI9 / Taq I2 primer.

도 4는 상기 도 2의 근내지방도 연관 139bp DNA 특이적 마커의 염기서열을 바탕으로 디자인된 프라이머를 이용하여 PCR을 수행한 결과이다.FIG. 4 shows the results of PCR using primers designed based on the nucleotide sequence of the 139 bp DNA-specific marker associated with intramuscular fat in FIG. 2.

본 발명은 1997년과 1999년 한우 육질평가대회 출품한우로부터 채취한 혈액을 원심분리하여 백혈구를 추출하고 단백질을 제거한 다음 에탄올로 DNA를 응축시키는 단계; 상기 DNA에TaqⅠ 제한 효소 (TaqⅠ restriction enzyme)와EcoRⅠ 제한 효소 (EcoRⅠ restriction enzyme)를 첨가하여 DNA 이중 침지 (double digestion)를 실시한 후 그 DNA 말단에 이중가닥 어댑터 (double strand adapter)를 라이게이션시키는 단계; AFLP (Amplified Fragment Length Polymorphism)의 PCR에 사용될 어댑터와 프라이머를 합성한 후 상기 준비한 DNA,TaqⅠ 프라이머,EcoRⅠ프라이머Taq폴리머라제 등을 혼합하여 PCR을 수행하는 단계; 상기 PCR 반응산물을 1% 아가로스 겔에서 전기영동시켜 반응 유무를 확인하고 그 겔을 굳힌 후 pre-run 시키는 단계; 상기 각 PCR 반응산물에 STR 3X 로딩 버퍼를 첨가하여 변성시킨 후 얼음에 꽂아 보관한 시약을 상기 전기영동한 플레이트의 각 well에 로딩한 후 전기영동하는 단계; 상기 플레이트를 고정 및 세척한 후 염색 및 현상하는 단계; 다형성 (polymorphism)을 가지는 특이적 DNA 마커를 아크릴아마이드 겔 상에서 잘라내어 상기와 동일한 프라이머로 PCR을 수행한 후 그 반응산물을 전기영동하여 단일밴드를 확인하고 염기서열을 분석하는 단계; 상기 염기서열을 바탕으로 하여 한우의 경제형질을 판정할 수 있는 프로브 및 프라이머를 디자인하여 본 발명 한우 근내지방도 연관 프라이머를 개발하는 단계 및 상기 제작한 근내지방도 연관 프라이머를 사용하여 한우의 육질을 검사하는 단계로 구성된다. 본 발명에서 개발한 한우의 육질을 검사할 수 있는 15mer의 프라이머의 염기배열의 일부를 다른 염기로 치환, 삭제하거나 일부 염기배열의 위치와 방향이 바뀐 염기서열도 본 발명 프라이머의 범위에 속할 것이다.The present invention comprises the steps of extracting white blood cells, removing proteins and condensing DNA with ethanol by centrifuging the blood collected from the Hanwoo meat quality assessment competition in 1997 and 1999; The Taq Ⅰ restriction enzyme (Taq Ⅰ restriction enzyme) and Eco RⅠ restriction enzymes (Eco RⅠ restriction enzyme) was added to the DNA double dipping the double-stranded adapter (double strand adapter) to the DNA ends and then subjected to a (double digestion) to the DNA Ligation; Synthesizing an adapter and a primer to be used for AFLP (Amplified Fragment Length Polymorphism) PCR, and then performing PCR by mixing the prepared DNA, Taq I primer, Eco R I primer Taq polymerase, and the like; Electrophoresis of the PCR reaction product on a 1% agarose gel to confirm the presence of the reaction, and after the gel is hardened, pre-run; Adding and denature the STR 3X loading buffer to each PCR reaction product, and loading the reagents stored in ice into each well of the electrophoretic plate, followed by electrophoresis; Fixing and washing the plate and then staining and developing the plate; Cutting out specific DNA markers having polymorphism on acrylamide gel and performing PCR with the same primers, followed by electrophoresis of the reaction product to identify single bands and analyzing nucleotide sequences; Designing a probe and a primer that can determine the economic traits of Hanwoo based on the base sequence to develop the present invention Hanwoo intramuscular fat associated primers and to examine the quality of Hanwoo beef using the prepared intramuscular fat associated primers It consists of steps. Part of the base sequence of the 15mer primer that can examine the meat quality of the Hanwoo developed in the present invention by substituting with another base, or changing the position and orientation of some base sequences will also fall within the scope of the primer of the present invention.

이하, 본 발명의 구체적인 방법을 실시예를 들어 상세히 설명하고자 하지만 본 발명의 권리범위는 이들에만 한정되는 것은 아니다.Hereinafter, specific examples of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited thereto.

실시예 1 : 본 발명 프라이머의 제조Example 1 Preparation of the Primer of the Invention

1997년과 1999년 한우 육질평가대회 출품우 168두 한우의 13번째 늑골과 첫 번째 요추를 절개한 배최장근 단면적의 근내지방도 (marbling score)형질을 대상으로 본 발명을 수행하였다.The present invention was performed on the marbling score of 168 long ribs of the Hanwoo meat quality assessment competition in 1997 and 1999.

제1단계. 한우 백혈구로부터 DNA의 추출First step. Extraction of DNA from Hanwoo Leukocytes

상기 본 발명 대상 한우의 백혈구로부터 DNA를 추출하였다.DNA was extracted from the leukocytes of the present invention.

0.5M EDTA가 처리된 주사기로 채취한 한우의 혈액을 0.2% NaCl 용액으로 용혈시킨 후 1000rpm에서 5분간 원심분리하여 백혈구층을 분리하고, 여기에 0.16M NaCl/1mM EDTA와 0.5% N-lauroylsarcosine용액, 그리고 Proteinase K (10㎎/㎖, Promeag Co. USA)를 처리하였다. 동일량의 TE : 페놀(phenol) (1:1)과 페놀 : 클로로포름(chloroform) : 이소-아밀알코올(Iso-amylalchol) (25:24:1)을 각각 1회씩 넣어 단백질을 제거시킨 후 3M 나트륨 아세테이트 [Sodium acetate (pH5.2)]를 첨가하고, 100% 에탄올을 넣어 DNA를 응축시켰다. 그 DNA를 TE 완충액 (10mM Tris-HCl pH8.0, 1mM EDTA)에 녹여 4℃에서 보관하였다.The blood of Hanwoo, collected with a 0.5M EDTA-treated syringe, was hemolysed with 0.2% NaCl solution and centrifuged at 1000 rpm for 5 minutes to separate the leukocyte layer, followed by 0.16M NaCl / 1mM EDTA and 0.5% N-lauroylsarcosine solution. And Proteinase K (10 mg / ml, Promeag Co. USA). The same amount of TE: phenol (1: 1) and phenol: chloroform: iso-amylalchol (25: 24: 1) was added once to remove protein, followed by 3M sodium Sodium acetate (pH5.2) was added, and 100% ethanol was added to condense DNA. The DNA was dissolved in TE buffer (10 mM Tris-HCl pH8.0, 1 mM EDTA) and stored at 4 ° C.

제2단계. DNA 이중 침지 (double digestion) 및 라이게이션 (ligation)Second step. DNA double digestion and ligation

상기 준비한 DNA 500ng에TaqⅠ 제한 효소 7.5 유닛 (TaqⅠ restriction enzyme 7.5 unit)을 첨가하여 총 부피 (total volume)가 25㎕가 되도록 한 후, 65℃에서 1시간동안 침지 (digestion)하였다. 그 후,EcoRⅠ 제한 효소 2.5 유닛을 첨가하여 총 부피가 50㎕가 되도록 하여 37℃에서 1시간동안 침지를 실시하였다.Was added to 7.5 units Taq Ⅰ restriction enzyme (Taq Ⅰ restriction enzyme 7.5 unit) in the prepared DNA 500ng total volume (total volume) such that 25㎕, it was immersed (digestion) for 1 hour at 65 ℃. Thereafter, 2.5 units of Eco Rl restriction enzyme were added to the total volume to 50 µl, and soaking was performed at 37 ° C for 1 hour.

상기 이중으로 침지한 DNA 절편 말단에 이중가닥 어댑터 (double stand adapter)를 라이게이션시키기 위하여 5 pmolTaqⅠ 이중가닥 어댑터, 5 pmolEcoRⅠ이중가닥 어댑터, 1mM ATP, 1 유닛 T4 DNA 리가아제 (ligase) 및 상기 이중 침지된 샘플 10㎕를 총 부피가 50㎕ 되도록 하여 37℃에서 3시간동안 라이게이션을 실시하였다. 라이게이션이 끝난 후, 희석 용액 (dilution solution; 10mM Tris-HCl, 0.1mM EDTA)을 460㎕ 첨가하여 희석시켰다.5 pmol Taq I double strand adapter, 5 pmol Eco Rl double strand adapter, 1 mM ATP, 1 unit T4 DNA ligase to ligate a double stand adapter to the double immersed DNA fragment ends 10 L of the double immersed sample was allowed to have a total volume of 50 L and then ligated at 37 ° C for 3 hours. After the ligation was completed, dilution solution (10 mM Tris-HCl, 0.1 mM EDTA) was added to dilute.

제3단계. PCR의 수행Third step. Perform PCR

AFLP (Vos등, 1995)의 PCR 반응에 사용될 어댑터와 프라이머는 Ajmone-Marsan등 (1997)과 Basedow등 (1998)이 축우 품종에 이미 사용하였던 어댑터 (adapter)와 프라이머 (primer)를 이용하여 Bioneer Co.(Korea)에서 합성하여 사용하였다. 그 어댑터와 프라이머의 염기서열을 표 1에 나타내었다.Adapters and primers to be used for the PCR reaction of AFLP (Vos et al., 1995) were used by Ajmone-Marsan et al. (1997) and Basedow et al. (1998). It was synthesized from. (Korea). The base sequences of the adapters and primers are shown in Table 1.

AFLP-PCR을 위한 어댑터와 프라이머의 DNA 염기서열DNA sequences of adapters and primers for AFLP-PCR 명칭designation 서열order 어댑터EcoRⅠAdapter EcoR I Ecotop strand Eco top strand 5′- CTCGTAGACTGCGTACC5′- CTCGTAGACTGCGTACC Ecobottom strand Eco bottom strand 5′- AATGGTACGCAGTCTAC5′- AATGGTACGCAGTCTAC 어댑터TaqⅠAdapter Taq Ⅰ Taqtop strand Taq top strand 5′- GACGATGAGTCCTGAC5′- GACGATGAGTCCTGAC Taqbottom strand Taq bottom strand 5′- CGGTCAGGACTCAT5′- CGGTCAGGACTCAT 프라이머EcoRⅠPrimer EcoR I EP 1EP 1 5′- GACTGCGTACCAATTCA5′- GACTGCGTACCAATTCA EP 2EP 2 5′- GACTGCGTACCAATTCAAC5′- GACTGCGTACCAATTCAAC EP 3EP 3 5′- GACTGCGTACCAATTCAAG5′- GACTGCGTACCAATTCAAG EP 4EP 4 5′- GACTGCGTACCAATTCACA5′- GACTGCGTACCAATTCACA EP 5EP 5 5′- GACTGCGTACCAATTCACT5′- GACTGCGTACCAATTCACT EP 6EP 6 5′- GACTGCGTACCAATTCAGA5′- GACTGCGTACCAATTCAGA EP 7EP 7 5′- GACTGCGTACCAATTCAGT5′- GACTGCGTACCAATTCAGT EP 8EP 8 5′- GACTGCGTACCAATTCATC5′- GACTGCGTACCAATTCATC EP 9EP 9 5′- GACTGCGTACCAATTCATG5′- GACTGCGTACCAATTCATG 프라이머TaqⅠPrimer Taq Ⅰ TP 1TP 1 5′- GATGAGTCCTGACCGAA5′- GATGAGTCCTGACCGAA TP 2TP 2 5′- GATGAGTCCTGACCGAAAC5′- GATGAGTCCTGACCGAAAC TP 3TP 3 5′- GATGAGTCCTGACCGAAAG5′- GATGAGTCCTGACCGAAAG TP 4TP 4 5′- GATGAGTCCTGACCGAACA5′- GATGAGTCCTGACCGAACA TP 5TP 5 5′- GATGAGTCCTGACCGAACT5′- GATGAGTCCTGACCGAACT TP 6TP 6 5′- GATGAGTCCTGACCGACAC5′- GATGAGTCCTGACCGACAC TP 7TP 7 5′- GATGAGTCCTGACCGACAG5′- GATGAGTCCTGACCGACAG TP 8TP 8 5′- GATGAGTCCTGACCGACAT5′- GATGAGTCCTGACCGACAT TP 9TP 9 5′- GATGAGTCCTGACCGACCA5′- GATGAGTCCTGACCGACCA [주] EP :EcoRⅠ프라이머TP :TaqⅠ프라이머EP: EcoR I Primer TP: Taq I Primer

침지와 라이게이션이 끝난 후, 희석시킨 상기 DNA 샘플 3㎕와 75ngTaqⅠ 프라이머, 75ngEcoRⅠ프라이머, 0.2mM dNTP, 1mM MgCl2,0.1배 10x 완충액 및 1 유닛 Taq 폴리머라제 (polymerase)를 혼합하여 총 부피가 25㎕가 되도록 멸균 증류수를 첨가하였다. 반응은 94℃에서 30초 (denaturation), 60℃에서 1분 (annealing), 72℃에서 1분 (extention)의 순서대로 35주기를 T-Gradient thermal cycler (Biometra, Germany)에서 수행한 뒤 72℃에서 10분간 1회를 수행하였다.After immersion and ligation, 3 μl of the diluted DNA sample was mixed with 75 ng Taq I primer, 75 ng Eco R I primer, 0.2 mM dNTP, 1 mM MgCl 2, 0.1-fold 10 × buffer and 1 unit Taq polymerase. Sterile distilled water was added to bring the total volume to 25 μl. The reaction was carried out in a T-Gradient thermal cycler (Biometra, Germany) after 35 cycles of 30 seconds (denaturation) at 94 ° C, 1 minute at 60 ° C, and 1 minute at 72 ° C, followed by 72 ° C. One run for 10 minutes at.

제4단계. 전기영동 (Electrophoresis)Fourth step. Electrophoresis

상기 DNA PCR 반응산물에 대하여 전기영동을 수행하였다.Electrophoresis was performed on the DNA PCR reaction product.

상기 PCR 산물을 1% 아가로스 겔에서 전기영동하여 반응의 유무를 확인하고, 6% denaturing polyacrylamide gel (6% acryamide solution, 7M urea, 1X TBE buffer)을 준비하여 TEMED (Biorad, USA)와 10% Ammonium persulfate를 첨가하여 겔을 굳히고, 겔 플레이트를 약 50℃가 될 때까지 pre-run시켜, 각각의 PCR 산물 6㎕에 STR 3X 로딩 완충액 (10mM NaOH, 95% formamide, 0.05% bromophenol blue, 0.05% xylene cyanol FF) 3㎕를 첨가하여 95℃에서 2분간 denaturating 시킨 후에 즉시 얼음에 꽂아두고, 그 중 6㎕를 각 well에 로딩하여, 1,900Volts에서 50mA, 75W로 3시간동안 SQ3 sequencer (Hoefer Pharmacia Biotech Igc., USA)에서 전기영동하였다.The PCR product was electrophoresed on a 1% agarose gel to confirm the presence of the reaction, and a 6% denaturing polyacrylamide gel (6% acryamide solution, 7Murea, 1X TBE buffer) was prepared and 10% with TEMED (Biorad, USA). Ammonium persulfate was added to solidify the gel and pre-run the gel plate to about 50 ° C. in 6 μl of each PCR product in STR 3X loading buffer (10 mM NaOH, 95% formamide, 0.05% bromophenol blue, 0.05% 3 μl of xylene cyanol FF) was added, denaturated at 95 ° C. for 2 minutes, immediately placed on ice, and 6 μl of which was loaded into each well. The SQ3 sequencer (Hoefer Pharmacia Biotech) Igc., USA).

제5단계. Silver staining5th step. Silver staining

상기 전기영동한 겔 플레이트를 고정 용액 (fix solution; 10% acetic acid)으로 20분간 고정시킨 후, 증류수로 2분간 3차례 세척하고, 30분간 염색 용액 (2g silvet nitrate, 3㎖ 37% formaldehyde)으로 염색한다. 약 3ℓ의 증류수로 10초간 세척한 후 developer solution (60g sodium carbonate, 3㎖ 37% formaldehyde, 400㎕-10㎎/㎖ sodium tiosulfate)으로 현상하고 고정 용액을 부어 상을 고정시킨 후 증류수로 2분간 세척하여 glass plate를 건조시켰다.The electrophoretic gel plate was fixed in a fixed solution (fix solution; 10% acetic acid) for 20 minutes, washed three times for 2 minutes with distilled water, and then dyed for 30 minutes with a dye solution (2 g silvet nitrate, 3 ml 37% formaldehyde). Dye After 10 seconds of washing with about 3 liters of distilled water, develop with developer solution (60g sodium carbonate, 3ml 37% formaldehyde, 400μl-10mg / ml sodium tiosulfate), pour fixed solution, fix phase and wash with distilled water for 2 minutes. The glass plate was dried.

그 결과, 근내지방도 성적이 높은 집단 (High group)에서 402bp 위치에서,낮은 집단 (Low group)에서 179bp 및 139bp의 위치에서 차별화되어 나타나는 DNA 마커를 확인할 수 있었다. 그 결과를 도 1에 나타내었다.As a result, DNA markers that were differentiated at 402 bp in the high group and 179 bp and 139 bp in the low group were identified. The results are shown in FIG.

제6단계. sequence의 분석Step 6. sequence analysis

상기 아크릴아마이드 겔로부터 분리한 근내지방도 성적이 낮은 집단에서 나타난 특이적 139bp DNA 마커의 염기서열을 분석하였다.The nucleotide sequence of the specific 139 bp DNA marker shown in the low intramuscular fat score isolated from the acrylamide gel was analyzed.

상기 다형성 (Polymorphism)을 가지는 402bp, 179bp, 및 139bp의 DNA 마커 (marker) 중 139bp DNA 마커를 아크릴아마이드 겔 (acrylamide gel) 상에서 잘라내어 동일 프라이머를 사용하여 PCR을 수행하고, PCR 산물을 1% 아가로스 겔 상에서 전기영동하여 단일 밴드를 확인하였다. QIA quickTMPCR Purification Kit (QIAGEN, Germany, Cat. No. 28104)로 얻고자 하는 순수한 산물을 가지고 ABI PRISM 377 automatic sequencer (Perkin-Elmer, USA)를 이용하여 sequencing을 수행한 결과 염기서열을 확인하였다. 그 염기서열 분석 결과를 도 2에 나타내었다.The 139 bp DNA markers of the 402 bp, 179 bp, and 139 bp DNA markers having the polymorphism were cut out on an acrylamide gel, and PCR was performed using the same primers.The PCR product was subjected to 1% agarose. Electrophoresis on the gel confirmed a single band. The sequencing was performed using ABI PRISM 377 automatic sequencer (Perkin-Elmer, USA) with the pure product to be obtained with QIA quick TM PCR Purification Kit (QIAGEN, Germany, Cat. No. 28104). . The sequencing results are shown in FIG. 2.

제7단계. 프라이머 및 프로브의 제작Step 7. Preparation of primers and probes

상기 확인된 서열 결과를 토대로 프라이머 디자인 프로그램을 이용하여 한우의 증체량과 근내지방도에 연관된 경제형질을 판정할 수 있는 고유한 반복 서열을 이용한 프로브나 GC 성분 50% 이상, 이차 구조 (secondary structure)가 없고, 녹는 점 [melting temperature (Tm)]이 50℃ 이상인 조건을 만족하도록 하여 정확한결과를 나타내는 프라이머를 디자인함으로서, 한우에서 고유하게 적용될 수 있는 빠르고 간편한 기술로 한우의 능력개량을 위한 수단으로 이용할 수 있도록 하였다. 상기EcoRⅠ9/TaqⅠ2 프라이머로 규명된 근내지방도에 연관된 139bp DNA 마커의 염기서열을 분석하여 제작한 프라이머를 도 3의 ① 및 ②에 나타내었다. 여기서 ①은 MS139L (forward primer; 15mer)를 나타내며 ②는 MS 139R (reverse primer; 15mer)를 나타낸다.50% or more of probe or GC components using a unique repeat sequence that can determine the economic traits related to the weight gain and intramuscular fat of Hanwoo using the primer design program based on the identified sequence results, and there is no secondary structure By designing primer that shows accurate results by satisfying the condition that melting point [melting temperature (Tm)] is over 50 ℃, it can be used as a means for improving the ability of Hanwoo by a quick and simple technology that can be applied uniquely in Hanwoo It was. The primers produced by analyzing the nucleotide sequence of the 139 bp DNA marker related to the intramuscular fat identified by the Eco RI9 / Taq I2 primer are shown in ① and ② of FIG. 3. Where ① represents MS139L (forward primer; 15mer) and ② represents MS 139R (reverse primer; 15mer).

실시예 2 : 상기 본 발명 프라이머를 이용한 한우 근내지방도 성적 검사의 실시Example 2 implementation of the Hanwoo muscle fat score test using the present invention primer

한우 DNA 시료를 추출한 후 상기 본 발명 프라이머를 이용한 일반적 PCR을 수행함으로써 한우 근내지방도 성적 검사를 실시하였다.Hanwoo DNA samples were extracted and subjected to general PCR using the primers of the present invention.

상기 139bp 마커의 서열에서 디자인된 forward primer AAGTGTGGCGGTGGA 및 reverse primer CGGACACTGTGCTTC 중 MS139R을 이용하여 분석대상개체 168두 전체에 대하여 PCR을 수행한 결과를 도 4에 나타내었으며 그 분석 결과를 표 2에 나타내었다.PCR of all 168 subjects using MS139R of the forward primer AAGTGTGGCGGTGGA and reverse primer CGGACACTGTGCTTC designed from the sequence of the 139 bp marker was shown in FIG. 4, and the analysis results are shown in Table 2.

한우의 근내지방도 성적 검사를 위한 MS139R 이용 특이적 DNA 마커의 분리Isolation of MS139R-Specific DNA Markers for Intramuscular Fat Test of Korean Cattle 밴드 있음In band 밴드 없음No band 총 (total)Total 3등급Grade 3 1 (3.1%)1 (3.1%) 1 (0.7%)1 (0.7%) 2 (1.2%)2 (1.2%) 2등급Grade 2 11 (34.4%)11 (34.4%) 27 (19.9%)27 (19.9%) 38 (22.6%)38 (22.6%) 1등급Grade 1 7 (21.9%)7 (21.9%) 37 (27.2%)37 (27.2%) 44 (26.2%)44 (26.2%) 1+등급1+ grade 13 (40.6%)13 (40.6%) 71 (52.2%)71 (52.2%) 84 (50.0%)84 (50.0%) 1등급 전체(1등급)+(1+등급)1st grade (1st grade) + (1+ grade) 20 (62.5%)20 (62.5%) 108 (79.4%)108 (79.4%) 128 (76.2%)128 (76.2%) total individuals (%)total individuals (%) 32 (100%)32 (100%) 136 (199%)136 (199%) 168 (100%)168 (100%)

상기 본 발명 프라이머 MS139L 및 MS139R을 이용하여 검사한 결과, 밴드를 가지고 있는 집단의 성적은 전체 1등급 출현이 62.5%를 나타내고 마커가 나타나지 않는 집단의 1등급 출현율은 79.4%를 나타내고 있어 전체적으로 17% 이상의 근내지방 능력을 높일 수 있는 새로운 프라이머의 개발이 이루어졌다고 판단된다. 이러한 디자인된 상기 본 발명 프라이머를 이용하면, 복잡한 단계를 거쳐 결과를 볼 수 있는 AFLP의 실험방법보다는 PCR로서 간단하고 빠르게 결과를 확인할 수 있으며 근내지방도를 미리 예측할 수 있는 매우 유용한 방법으로 이용될 수 있다.As a result of testing using the primers MS139L and MS139R of the present invention, the results of the group having the band showed 62.5% of the total grade 1 appearance, and the grade 1 appearance rate of the group without the marker showed 79.4%, and the overall result was 17% or more. We believe that a new primer has been developed to increase muscle fat. Using the designed primer of the present invention, it is possible to check the result simply and quickly as PCR rather than the experimental method of AFLP which can see the result through a complicated step, and can be used as a very useful method for predicting intramuscular fat in advance. .

이상의 실시예를 통하여 명백한 바와 같이, 본 발명은 한우의 genomic DNA를 함성하여 제조한 AFLPEcoRⅠ9/TaqⅠ2 프라이머와 반응시켜 근내지방도 성적이 높은 집단과 낮은 집단에서 차별화되는 DNA 마커의 염기서열을 분석하고 그로부터 한우의 주요 형질, 특히 한우의 근내지방도와 연관된 프라이머와 프로브를 개발하여 제공하는 효과가 있다. 또한, 그 프라이머를 이용하여 한우 발육 단계에 관계없이근내지방도가 우수하고 육질이 뛰어난 비육우를 보다 바람직하고 손쉽고 간편하게 판별할 수 있는 한우 육질, 특히 근내지방도 성적 검사방법을 제공하는 뛰어난 효과가 있으므로, 축산업상 매우 유용한 발명인 것이다.As is apparent from the above examples, the present invention is to analyze the nucleotide sequence of DNA markers that are differentiated in the high and low myopia scores by reacting with the AFLP Eco R9 / Taq I2 primer prepared by the genomic DNA of Hanwoo From there, it is effective to develop and provide primers and probes related to the major traits of Korean cattle, especially the intramuscular fat of Korean cattle. In addition, since the use of the primer has excellent effect of providing a test method for the quality of the beef, especially the intramuscular fat, which makes it possible to discriminate more preferably, easily and easily, beef cattle having excellent intramuscular fat and excellent meat quality regardless of the stage of Hanwoo development. It is a very useful invention.

Claims (3)

하기와 같은 한우 (Hanwoo) 유전자 유래의 근내지방도 연관 특이적 DNA 표지인자 염기서열.Specific DNA marker sequences associated with intramuscular fat derived from Hanwoo gene as follows. 상기 제 1항 기재의 한우 유전자 유래의 근내지방도 연관 특이적 DNA 표지인자 염기서열을 분석하여 제작한 하기 서열의 한우 근내지방도 판별 프라이머 MS139L 또는 MS 139R.MS139L or MS 139R of Hanwoo muscle fat determination primer of the following sequence prepared by analyzing the specific DNA marker nucleotide sequence related to myocardial fat derived from the Hanwoo gene of claim 1. 삭제delete
KR10-2000-0065925A 2000-11-07 2000-11-07 Primer related marbling score of Hanwoo and test process of marbling score using thereof KR100415080B1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
KR10-2000-0065925A KR100415080B1 (en) 2000-11-07 2000-11-07 Primer related marbling score of Hanwoo and test process of marbling score using thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
KR10-2000-0065925A KR100415080B1 (en) 2000-11-07 2000-11-07 Primer related marbling score of Hanwoo and test process of marbling score using thereof

Publications (2)

Publication Number Publication Date
KR20020035678A KR20020035678A (en) 2002-05-15
KR100415080B1 true KR100415080B1 (en) 2004-01-13

Family

ID=19697712

Family Applications (1)

Application Number Title Priority Date Filing Date
KR10-2000-0065925A KR100415080B1 (en) 2000-11-07 2000-11-07 Primer related marbling score of Hanwoo and test process of marbling score using thereof

Country Status (1)

Country Link
KR (1) KR100415080B1 (en)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007100578A2 (en) * 2006-02-22 2007-09-07 Washington State University Research Foundattion A simplified qtl mapping approach for screening and mapping novel markers associated with beef marbling
WO2008061175A3 (en) * 2006-11-15 2009-02-05 Univ Washington Polymorphisms in the urocortin 3 gene and their associations with marbling and subcutaneous fat depth in beef cattle
KR100938634B1 (en) 2008-02-19 2010-01-22 고려대학교 산학협력단 A prediction method of pork quality traits using blood glucose
KR100961677B1 (en) 2007-09-07 2010-06-09 건국대학교 산학협력단 Method for detecting cattle with high marbling

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100590728B1 (en) * 2004-11-11 2006-06-19 영남대학교 산학협력단 Microsatellite dna associated with economic traits in hanwoo and primer for detecting same
KR100804310B1 (en) * 2006-07-13 2008-02-18 대한민국 4 DNA marker of adipocyte-fatty acid binding protein gene related the intramuscular fat content in beef cattle
CN107746884B (en) * 2017-11-29 2020-09-29 中国农业科学院农业质量标准与检测技术研究所 AFLP primer combination product, kit and method for identifying individual and variety of beef cattle

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR970007377A (en) * 1995-07-13 1997-02-21 문정환 Manual Sockets for Testing Electrical Characteristics of Semiconductor Devices
WO1999023248A1 (en) * 1997-10-30 1999-05-14 Commonwealth Scientific And Industrial Research Organisation Assessing lipid metabolism
KR100282246B1 (en) * 1998-02-25 2001-04-02 김강권 Beef quality related DNA markers and selection method of bovine sarcoma

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR970007377A (en) * 1995-07-13 1997-02-21 문정환 Manual Sockets for Testing Electrical Characteristics of Semiconductor Devices
WO1999023248A1 (en) * 1997-10-30 1999-05-14 Commonwealth Scientific And Industrial Research Organisation Assessing lipid metabolism
KR100282246B1 (en) * 1998-02-25 2001-04-02 김강권 Beef quality related DNA markers and selection method of bovine sarcoma

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
논문(청구항 1항 서열에 대한 BLAST 검색결과[유전자 서열코드 AF297628 첨부,2001.7.11) *
논문(청구항 2항 프라이머에 대한 BLAST 검색결과,2001.7.11) *
논문(한우 육질 개선을 위한 검정 프라이머 및 염기서열[46, 79쪽 서열] [과학기술부 1997년 특정연구개발서업 보고서 ''유전공학기법에 의한 가축개량 기술개발 연구,1998.7.30) *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007100578A2 (en) * 2006-02-22 2007-09-07 Washington State University Research Foundattion A simplified qtl mapping approach for screening and mapping novel markers associated with beef marbling
WO2007100578A3 (en) * 2006-02-22 2008-10-16 Univ Washington A simplified qtl mapping approach for screening and mapping novel markers associated with beef marbling
WO2008061175A3 (en) * 2006-11-15 2009-02-05 Univ Washington Polymorphisms in the urocortin 3 gene and their associations with marbling and subcutaneous fat depth in beef cattle
KR100961677B1 (en) 2007-09-07 2010-06-09 건국대학교 산학협력단 Method for detecting cattle with high marbling
KR100938634B1 (en) 2008-02-19 2010-01-22 고려대학교 산학협력단 A prediction method of pork quality traits using blood glucose

Also Published As

Publication number Publication date
KR20020035678A (en) 2002-05-15

Similar Documents

Publication Publication Date Title
US7598061B2 (en) Mold infections
CN113481286B (en) MiRNA-208a amplification primer pair based on strand exchange amplification and detection kit thereof
KR100415080B1 (en) Primer related marbling score of Hanwoo and test process of marbling score using thereof
KR100769366B1 (en) Ssr primer derived from mungbean and use thereof
KR100842432B1 (en) Ssr primer derived from mandarin and use thereof
CN112322768B (en) Rapid RPA detection method for diagnosing sea-buckthorn branch blight and pathogenic bacteria
KR20050046330A (en) Method discrimination for product traceability and identification of beef meat
KR20200017196A (en) Primer set for multiple detection apple and pear viruses and viroid and method for detecting said viruses and viroid using the same
US5674687A (en) Method for identifying the species origin of a DNA sample
KR101735075B1 (en) Composition and method for prediction of swine fecundity using genomic differentially methylated region
KR100653400B1 (en) Method for Distinguishing Korean Beef from Non-Korean Beef and Probe and Primer therefor
CN113355441B (en) Kit, primer pair, probe and method for detecting Sarcocystis
KR101006079B1 (en) SSR primer derived from Garlic and use of there
CN100503839C (en) Method for detecting CVM deleterious gene of oxen
KR102212379B1 (en) Primer set for simultaneous detection of three viruses in Grapevine and method for detecting three viruses in Grapevine using the same
CN111719020A (en) Kit, primer and probe for detecting bovine rotavirus
KR100842429B1 (en) Ssr primer derived from lawn grass and use thereof
KR100769367B1 (en) Ssr primer derived from common millet and use thereof
KR102336624B1 (en) Marker for predicting collagen content in pork and use thereof
CN112501310B (en) LAMP (loop-mediated isothermal amplification) detection primer group, kit and method for bovine-derived components
TWI268959B (en) Method for meat identification using mitochondrial 12S rRNA of cattle, goat or pig specific primers
CN117925860B (en) SNP molecular marker related to growth traits of puffer green and application thereof
KR102319859B1 (en) Multiplex primer sets based on microsatellite markers for identification of Korean native chicken and uses thereof
WO2024123125A1 (en) Single nucleic acid base polymorph marker for determining origin of pagus major, and use thereof
KR101337668B1 (en) DNA fragment markers for detecting increase of porcine Myosin heavy chain slow isoform and improvement on pork quality

Legal Events

Date Code Title Description
A201 Request for examination
N231 Notification of change of applicant
E902 Notification of reason for refusal
E701 Decision to grant or registration of patent right
N231 Notification of change of applicant
GRNT Written decision to grant
FPAY Annual fee payment

Payment date: 20121119

Year of fee payment: 10

FPAY Annual fee payment

Payment date: 20131011

Year of fee payment: 11

LAPS Lapse due to unpaid annual fee