KR20230123623A - Positive control plasmid for screening genetically modified canola and method for screening genetically modified canola using the same - Google Patents
Positive control plasmid for screening genetically modified canola and method for screening genetically modified canola using the same Download PDFInfo
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Abstract
Description
본 발명은 유전자변형 유채를 검정하기 위한 양성대조군 플라스미드 및 이를 이용한 유전자변형 유채의 검정방법에 관한 것이다.The present invention relates to a positive control plasmid for assaying genetically modified rapeseed and a method for assaying genetically modified rapeseed using the same.
유전자변형 생물체(Living Modified Organism, LMO)는 인류가 직면한 식량부족, 환경변화, 대체에너지 개발 등 각종 문제점의 해결방안으로, 고부가가치 창출이 가능하여 다양한 작물에 대하여 연구 개발이 진행되어 왔다. 유전자변형 토마토가 처음으로 개발되어 상업화된 이후 다양한 작물에 대한 LMO가 개발되어 왔으며 현재는 의약품, 기능성 등의 가치를 지니는 LMO 개발로 확대되고 있다. 2008년 '유전자변형생물체의 국가간 이동에 관한 법률(LMO법)'이 시행됨에 따라 LMO 수입의 용도별 수입 승인이 소관부처별로 의무화되고 국가차원에서 사후관리 등의 조치를 취하고 있다. 또한, LMO 원료 및 생산물의 안전성 확보와 소비자의 알권리 제공의 일환으로 환경위해성 및 인체안전성 평가를 의무화하고 있다. Living Modified Organism (LMO) is a solution to various problems faced by mankind, such as food shortage, environmental change, and development of alternative energy, and research and development has been conducted on various crops because it can create high added value. Since genetically modified tomatoes were first developed and commercialized, LMOs for various crops have been developed, and are now expanding to development of LMOs with value such as medicines and functionalities. In 2008, as the 'Act on Transboundary Movement of Living Modified Organisms (LMO Act)' was enforced, import approval for each use of LMO imports became mandatory for each department in charge, and measures such as follow-up management were taken at the national level. In addition, as part of securing the safety of LMO raw materials and products and providing consumers with the right to know, environmental risk and human safety assessments are mandated.
최근들어 미승인 LMO의 불법생산, 불법유통 또는 환경방출(environmental release)이 국가적인 이슈가 되고 있는 상황에서, LMO 작물의 재배 및 유통 관리와 소비자의 안전성 확보를 위하여 신속하고 효율적인 LMO 검출 기법에 대한 개발이 요구되고 있다. 특히 최근 개발되는 대부분의 LMO는 후대 교배종으로, 이에 대한 신속한 검출방법의 필요성이 증가되고 있다. 현재 LMO를 검출할 수 있는 방법으로는 단백질의 항원항체반응을 이용한 스트립(strip) 및 ELISA(Enzyme-Linked Immunosorbent Assay) 분석법이 있으며, 보다 정확하고 안정적으로 검출할 수 있는 PCR(Polymerase Chain Reaction) 분석법이 있다. In a situation where the illegal production, illegal distribution or environmental release of unapproved LMO has recently become a national issue, development of a fast and efficient LMO detection technique to manage the cultivation and distribution of LMO crops and to secure consumer safety. this is being requested In particular, most recently developed LMOs are progeny hybrids, and the need for rapid detection methods for them is increasing. Currently, methods that can detect LMO include strip and ELISA (Enzyme-Linked Immunosorbent Assay) assays using antigen-antibody reactions of proteins, and PCR (Polymerase Chain Reaction) assays that can detect more accurately and stably. there is
LMO의 모니터링을 위해서는 다양한 이벤트(계통)에 대한 검출 여부를 확인해야 한다. 이때 각 도입 유전자별 분석을 수행하여야 하나, 후대교배종 LMO의 경우 2~5개의 유전자가 집적되어 있어, 이들 유전자의 개별적 분석을 해야 하는 불편함이 있다. 또한, 비의도적 환경방출이 우려되는 상황에서 알려져 있지 않는 수많은 LMO 이벤트들을 모두 검출하기에는 많은 시간과 비용이 소모된다. 또한, 양성 시료는 구매가 어렵거나 고가이며, 반복 사용으로 인한 오염 문제가 우려되고 있으므로, 클론 형태의 양성대조군 확보가 요구된다.For LMO monitoring, it is necessary to check whether various events (systems) are detected. At this time, analysis should be performed for each transgene, but in the case of the LMO of the progeny hybrid, 2 to 5 genes are integrated, which is inconvenient to individually analyze these genes. In addition, it takes a lot of time and money to detect all the many unknown LMO events in a situation where unintentional environmental release is a concern. In addition, positive samples are difficult or expensive to purchase, and there is a concern about contamination due to repeated use, so it is required to secure a positive control in the form of a clone.
한편, 한국등록특허 제1739472호에 Topas 19/2, Rf3, Ms8, RT73, 및 T45의 '캐놀라 이벤트를 선별하기 위한 프라이머 세트, 방법 및 키트'가 개시되어 있으나, 본 발명의 Ms8, Rf3, GT73(RT73), T45, Ms1, Rf1, Rf2, Topas19-2, Mon88302, DP-073496-4, Ms11, LBFLFK 또는 NS-B50027-4의 캐놀라 이벤트를 선별할 수 있는 '유전자변형 유채를 검정하기 위한 양성대조군 플라스미드 및 이를 이용한 유전자변형 유채의 검정방법'에 대해서는 기재된 바가 없다.Meanwhile, Korean Patent Registration No. 1739472 discloses a 'primer set, method and kit for selecting canola events' of Topas 19/2, Rf3, Ms8, RT73, and T45, but Ms8, Rf3, GT73 of the present invention (RT73), T45, Ms1, Rf1, Rf2, Topas19-2, Mon88302, DP-073496-4, Ms11, LFBLFK or NS-B50027-4 to screen for canola events of 'positive to test transgenic rape' There is nothing described about the control plasmid and the assay method of genetically modified rape using the control plasmid.
본 발명은 상기와 같은 요구에 의해 도출된 것으로서, 본 발명자들은 유전자변형 유채 계통인 Ms8, Rf3, GT73(RT73), T45, Ms1, Rf1, Rf2, Topas19-2, Mon88302, DP-073496-4, Ms11, LBFLFK(1), LBFLFK(2), NS-B50027-4(1) 및 NS-B50027-4(2)와 유채 내재 유전자인 CruA(Cruciferin A)에 각각 특이적인 프라이머 세트의 염기서열을 포함하는 양성대조군 플라스미드를 제작하였다. 또한, 상기 양성대조군 서열을 주형으로 이용하여 PCR을 수행한 결과, 양성대조군 서열의 제작에 사용된 각각의 프라이머 세트(표 1)가 비특이적 반응없이 예상되는 크기의 PCR 산물을 증폭하는 것을 확인하였고, 이를 통해 본 발명의 양성대조군 플라스미드는 기존 PCR 검사법을 이용한 유전자변형 유채 검정 시 양성대조군으로 효과적으로 기능할 수 있음을 확인함으로써, 본 발명을 완성하였다.The present invention has been derived from the above needs, and the present inventors have genetically modified rapeseed lines Ms8, Rf3, GT73 (RT73), T45, Ms1, Rf1, Rf2, Topas19-2, Mon88302, DP-073496-4, Ms11, LBFLFK(1), LBFLFK(2), NS-B50027-4(1) and NS-B50027-4(2) and the nucleotide sequence of primer sets specific for CruA (Cruciferin A), an endogenous rapeseed gene, are included. A positive control plasmid was prepared. In addition, as a result of performing PCR using the positive control sequence as a template, it was confirmed that each primer set (Table 1) used in the preparation of the positive control sequence amplified a PCR product of the expected size without nonspecific reaction, Through this, the present invention was completed by confirming that the positive control plasmid of the present invention can effectively function as a positive control during genetically modified rapeseed assay using an existing PCR test method.
상기 과제를 해결하기 위해, 본 발명은 서열번호 1의 염기서열로 이루어진 유전자변형 유채 식물체 검정용 양성대조군 플라스미드를 제공한다.In order to solve the above problems, the present invention provides a positive control plasmid for assaying transgenic rape plants consisting of the nucleotide sequence of SEQ ID NO: 1.
또한, 본 발명은 서열번호 1의 염기서열로 이루어진 양성대조군 플라스미드를 포함하는 유전자변형 유채 식물체 검정용 플라스미드 조성물을 제공한다.In addition, the present invention provides a plasmid composition for assaying transgenic oilseed rape plants comprising a positive control plasmid consisting of the nucleotide sequence of SEQ ID NO: 1.
또한, 본 발명은 서열번호 1의 염기서열로 이루어진 양성대조군 플라스미드를 포함하는 유전자변형 유채 식물체 검정용 키트를 제공한다.In addition, the present invention provides a kit for assaying transgenic oilseed rape plants comprising a positive control plasmid consisting of the nucleotide sequence of SEQ ID NO: 1.
또한, 본 발명은 유전자변형 유채 시료의 핵산을 주형으로 하고, 서열번호 1의 염기서열로 이루어진 양성대조군 플라스미드를 양성대조군으로 이용하여 유전자변형 유채 식물체의 특이적 염기서열을 증폭하는 PCR 프라이머 세트를 이용하여 표적 핵산을 증폭하는 단계를 포함하는 유전자변형 유채 식물체를 검정하는 방법을 제공한다.In addition, the present invention uses the nucleic acid of the genetically modified rapeseed sample as a template and a positive control plasmid consisting of the nucleotide sequence of SEQ ID NO: 1 as a positive control to amplify the specific nucleotide sequence of the genetically modified rapeseed plant Using a PCR primer set It provides a method for assaying a genetically modified rapeseed plant comprising the step of amplifying a target nucleic acid.
본 발명의 양성대조군 플라스미드는 기존의 PCR 방법을 바탕으로 하여 유전자변형 유채 검출의 효율성을 높일 수 있으므로, 시중에서 유통 중이거나 수입되는 농산물 및 식품 등에서 다양한 형태의 유전자변형 유채 시료를 검출하는데 소요되는 시간, 비용 및 노동력을 절감할 수 있을 것이다.The positive control plasmid of the present invention can increase the efficiency of genetically modified rapeseed detection based on the existing PCR method, so the time required to detect various types of genetically modified rapeseed samples in agricultural products and foods distributed or imported on the market , cost and labor will be reduced.
도 1 내지 도 4는 8개 농도(10 ng, 5 ng, 2.5 ng, 1.25 ng, 0.625 ng, 0.3125 ng, 0.15625 ng, 0.078125 ng)의 양성대조군 서열(서열번호 1)을 주형으로 이용하여 PCR을 수행한 결과이다. 1 to 4 are 8 concentrations (10 ng, 5 ng, 2.5 ng, 1.25 ng, 0.625 ng, 0.3125 ng, 0.15625 ng, 0.078125 ng) of PCR using a positive control sequence (SEQ ID NO: 1) as a template is the result of doing
본 발명의 목적을 달성하기 위하여, 본 발명은 서열번호 1의 염기서열로 이루어진 유전자변형 유채 식물체 검정용 양성대조군 플라스미드를 제공한다.In order to achieve the object of the present invention, the present invention provides a positive control plasmid for assaying transgenic rape plants consisting of the nucleotide sequence of SEQ ID NO: 1.
본 발명에서 용어 '유전자변형 유채'는 자연상태의 생리적 증식·재조합 또는 전통적인 교배·선발에서 사용되지 않는 현대생명공학기술을 이용하여 생물종의 유전물질을 인위적으로 변형시킨 LMO(Living modified organisms) 유채를 의미한다.In the present invention, the term 'genetically modified rapeseed' refers to LMO (Living modified organisms) rapeseed artificially modified with the genetic material of a species using modern biotechnology, which is not used in natural physiological growth/recombination or traditional breeding/selection. means
본 발명은 또한, 서열번호 1의 염기서열로 이루어진 양성대조군 플라스미드를 포함하는 유전자변형 유채 식물체 검정용 양성대조군 플라스미드 조성물을 제공한다.The present invention also provides a positive control plasmid composition for assaying transgenic rape plants comprising a positive control plasmid consisting of the nucleotide sequence of SEQ ID NO: 1.
상기 서열번호 1의 양성대조군 플라스미드는 다양한 유전자변형 유채 계통과 유채 내재 유전자에 특이적인 프라이머 세트의 염기서열(표 1 참고)에 대한 표적 부위를 포함하여 인공적으로 합성한 것으로, 비특이적 반응없이 각 프라이머 세트의 예측되는 증폭 산물 크기와 동일한 크기의 증폭 산물을 생산할 수 있도록 합성되었다.The positive control plasmid of SEQ ID NO: 1 is artificially synthesized by including target sites for the nucleotide sequences (see Table 1) of primer sets specific to various genetically modified rapeseed lines and rapeseed endogenous genes, and each primer set without non-specific reaction. It was synthesized to produce an amplification product of the same size as the predicted amplification product size of .
구체적으로, 상기 서열번호 1의 염기서열로 이루어진 양성대조군 플라스미드는 유전자변형 유채 계통인 Ms8, Rf3, GT73(RT73), T45, Ms1, Rf1, Rf2, Topas19-2, Mon88302, DP-073496-4, Ms11, LBFLFK(1), LBFLFK(2), NS-B50027-4(1) 및 NS-B50027-4(2)와 유채 내재 유전자인 CruA(Cruciferin A)에 각각 특이적인 총 16개의 프라이머 세트에 대한 표적 부위의 염기서열과 인위적인 염기서열을 추가하여 인공적으로 합성한 염기서열로, 서열번호 1의 양성대조군 플라스미드는 표 1에 개시된 총 16개의 프라이머 세트에 대한 양성대조군으로 기능할 수 있다.Specifically, the positive control plasmid consisting of the nucleotide sequence of SEQ ID NO: 1 is genetically modified rape strains Ms8, Rf3, GT73 (RT73), T45, Ms1, Rf1, Rf2, Topas19-2, Mon88302, DP-073496-4, For a total of 16 primer sets specific to Ms11, LBFLFK(1), LBFLFK(2), NS-B50027-4(1) and NS-B50027-4(2) and the rape endogenous gene CruA (Cruciferin A), respectively As a nucleotide sequence artificially synthesized by adding the nucleotide sequence of the target site and the artificial nucleotide sequence, the positive control plasmid of SEQ ID NO: 1 can function as a positive control for a total of 16 primer sets disclosed in Table 1.
본 발명은 또한, 서열번호 1의 염기서열로 이루어진 양성대조군 플라스미드를 포함하는 유전자변형 유채 식물체 검정용 키트를 제공한다.The present invention also provides a kit for assaying transgenic rapeseed plants comprising a positive control plasmid consisting of the nucleotide sequence of SEQ ID NO: 1.
본 발명의 키트에서, 상기 유전자변형 유채 식물체 검정용 양성대조군 플라스미드는 전술한 것과 같다.In the kit of the present invention, the positive control plasmid for assaying the genetically modified rapeseed plant is as described above.
상기 키트는 구체적으로, 서열번호 1의 염기서열로 이루어진 양성대조군 플라스미드, 유전자변형 유채 특이적 프라이머 세트, 및 증폭반응을 수행하기 위한 시약을 포함할 수 있으나, 이에 제한되지 않는다.Specifically, the kit may include, but is not limited to, a positive control plasmid consisting of the nucleotide sequence of SEQ ID NO: 1, a genetically modified rape-specific primer set, and reagents for performing an amplification reaction.
또한, 본 발명의 키트는 유전자변형 유채를 검정할 수 있는 프라이머 세트를 포함한다. 본 발명의 일 구현 예에 따른 상기 프라이머 세트는 표 1에 개시된 프라이머 세트일 수 있으나, 이에 제한되지 않는다.In addition, the kit of the present invention includes a primer set capable of assaying genetically modified rapeseed. The primer set according to one embodiment of the present invention may be a primer set disclosed in Table 1, but is not limited thereto.
본 발명의 일 구현 예에 있어서, 상기 증폭 반응을 수행하기 위한 시약은 DNA 폴리머라제, dNTPs 및 버퍼를 포함할 수 있으나, 이에 제한되지 않는다.In one embodiment of the present invention, reagents for performing the amplification reaction may include DNA polymerase, dNTPs, and buffers, but are not limited thereto.
본 발명의 일 구현 예에 있어서, 상기 키트는 또한 최적의 반응 수행 조건을 기재한 사용자 안내서를 추가로 포함할 수 있다. 안내서는 키트 사용법, 예를 들면, 역전사 완충액 및 PCR 완충액 제조 방법, 제시되는 반응 조건 등을 설명하는 인쇄물이다. 안내서는 팜플렛 또는 전단지 형태의 안내 책자, 키트에 부착된 라벨, 및 키트를 포함하는 패키지의 표면상에 설명을 포함한다. 또한, 안내서는 인터넷과 같이 전기 매체를 통해 공개되거나 제공되는 정보를 포함한다.In one embodiment of the present invention, the kit may further include a user guide describing optimal reaction performance conditions. The guide is a printout that explains how to use the kit, for example, how to prepare reverse transcription buffer and PCR buffer, and the reaction conditions to be presented. The guide includes a brochure in the form of a pamphlet or leaflet, a label affixed to the kit, and instructions on the surface of the package containing the kit. In addition, the guide includes information disclosed or provided through electronic media such as the Internet.
본 발명은 또한, 유전자변형 유채 시료의 핵산을 주형으로 하고, 서열번호 1의 염기서열로 이루어진 양성대조군 플라스미드를 양성대조군으로 이용하여 유전자변형 유채 식물체의 특이적 염기서열을 증폭하는 PCR 프라이머 세트를 이용하여 표적 핵산을 증폭하는 단계를 포함하는 유전자변형 유채 식물체를 검정하는 방법을 제공한다.The present invention also uses a nucleic acid of a genetically modified rapeseed sample as a template and a positive control plasmid consisting of the nucleotide sequence of SEQ ID NO: 1 as a positive control to amplify a specific nucleotide sequence of a genetically modified rapeseed plant Using a PCR primer set It provides a method for assaying a genetically modified rapeseed plant comprising the step of amplifying a target nucleic acid.
상기 시료는 유전자변형 유채와 관련된 식물, 식물 종자, 식물 세포, 후대 식물, 농업 생산물 등일 수 있으나, 이에 제한되지 않는다.The sample may be a plant, plant seed, plant cell, progeny plant, agricultural product, etc. related to genetically modified rapeseed, but is not limited thereto.
본 발명의 일 구현 예에 따른 방법에 있어서, 상기 서열번호 1의 염기서열로 이루어진 유전자변형 유채 식물체 검정용 양성대조군 플라스미드는 PCR 방법을 이용하여 유전자변형 유채를 검정하는 과정에서 Ms8, Rf3, GT73(RT73), T45, Ms1, Rf1, Rf2, Topas19-2, Mon88302, DP-073496-4, Ms11, LBFLFK(1), LBFLFK(2), NS-B50027-4(1) 또는 NS-B50027-4(2) 유전자변형 유체 계통을 특이적으로 검출할 수 있는 프라이머 세트에 대한 양성대조군으로 이용 가능하다.In the method according to one embodiment of the present invention, the positive control plasmid for genetically modified rape plant assay consisting of the nucleotide sequence of SEQ ID NO: 1 is Ms8, Rf3, GT73 ( RT73), T45, Ms1, Rf1, Rf2, Topas19-2, Mon88302, DP-073496-4, Ms11, LBFLFK(1), LBFLFK(2), NS-B50027-4(1) or NS-B50027-4( 2) It can be used as a positive control for a primer set that can specifically detect genetically modified fluid strains.
본 발명의 일 구현 예에 있어서, 상기 유전자변형 유채 식물체를 검정하는 방법은 유전자변형 유채 식물체의 핵산을 주형으로 하고, 각 유전자변형 유채의 특이적 염기서열을 증폭하는 단계를 통해 이루어질 수 있다. 상기 염기서열의 증폭은 당업계에 공지된 방법을 통해 수행될 수 있다.In one embodiment of the present invention, the method for assaying the genetically modified rapeseed plant may be performed by using the nucleic acid of the genetically modified rapeseed plant as a template and amplifying the specific nucleotide sequence of each genetically modified rapeseed. Amplification of the nucleotide sequence may be performed through a method known in the art.
이하, 본 발명을 실시예에 의해 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail by examples. However, the following examples are only to illustrate the present invention, and the content of the present invention is not limited to the following examples.
재료 및 방법Materials and Methods
1. 양성대조군 클론을 제작하기 위한 염기서열 설계 및 검증1. Base sequence design and verification to create positive control clones
본 발명자는 유전자변형(LMO) 유채 계통별 프라이머 세트들의 PCR 검사 시 양성대조군으로 사용될 수 있는 유채 양성대조군 서열(이하, 유채 양성대조군 클론)을 제작하였다. 상기 양성대조군 클론은, 본 발명의 LMO 유채 15계통 및 내재 유전자(CruA)에 각각 특이적인 총 16개의 프라이머 세트가 각각 비특이적 반응 없이 각 프라이머 세트의 예측되는 증폭 산물 크기와 동일한 크기의 증폭산물을 생산할 수 있도록 프라이머 염기서열과 인위적인 염기서열을 추가하여 설계한 염기서열(서열번호 1)이다(표 1). 본 발명에서는 양성대조군 염기서열을 설계한 후, pUC-GW-Amp 벡터(Genewiz, Inc.)에 클로닝된 형태로 준비하였다.The present inventors produced a positive control sequence (hereinafter, a positive control clone of rapeseed) that can be used as a positive control in the PCR test of primer sets for each genetically modified (LMO) rapeseed line. In the positive control clone, a total of 16 primer sets each specific to the LMO rapeseed 15 strains and endogenous gene ( CruA ) of the present invention produce amplification products of the same size as the expected amplification product size of each primer set without nonspecific reaction, respectively. It is a base sequence (SEQ ID NO: 1) designed by adding a primer base sequence and an artificial base sequence so that it can be used (Table 1). In the present invention, after designing the positive control nucleotide sequence, it was prepared in the form of cloning into the pUC-GW-Amp vector (Genewiz, Inc.).
구체적으로, 양성대조군 클론은 Ms8, Rf3, GT73(RT73), T45, Ms1, Rf1, Rf2, Topas19-2, Mon88302, DP-073496-4, Ms11, LBFLFK(1), LBFLFK(2), NS-B50027-4(1) 및 NS-B50027-4(2) 유전자변형 유채 계통과 유채 내재 유전자인 CruA(Cruciferin A)에 특이적인 총 16개의 프라이머 세트의 염기서열과 인위적인 염기서열을 추가하여 합성한 염기서열이다.Specifically, the positive control clones were Ms8, Rf3, GT73 (RT73), T45, Ms1, Rf1, Rf2, Topas19-2, Mon88302, DP-073496-4, Ms11, LBFLFK (1), LBFLFK (2), NS- B50027-4(1) and NS-B50027-4(2) bases synthesized by adding artificial base sequences and nucleotide sequences of a total of 16 primer sets specific to the genetically modified rapeseed line and CruA (Cruciferin A), an endogenous rapeseed gene. is a sequence
2. PCR(Polymerase Chain Reaction)2. PCR (Polymerase Chain Reaction)
유채 양성대조군 클론의 초기 농도를 10 ng으로 하여, 2배씩 희석하는 방식으로 8개의 농도(10 ng, 5 ng, 2.5 ng, 1.25 ng, 0.625 ng, 0.3125 ng, 0.15625 ng, 0.078125 ng)를 만들었다. PCR에 사용한 16개의 프라이머 세트는 LMO 유채 계통별 특이적 프라이머 세트와 내재 참조 유전자(CruA) 증폭용 프라이머 세트로 구성되었다.Eight concentrations (10 ng, 5 ng, 2.5 ng, 1.25 ng, 0.625 ng, 0.3125 ng, 0.15625 ng, 0.078125 ng) were made by diluting by 2 times with the initial concentration of rapeseed positive control clone being 10 ng. The 16 primer sets used for PCR consisted of a primer set specific to each LMO rapeseed line and a primer set for amplification of the endogenous reference gene ( CruA ).
PCR 과정은 전변성 95℃ 15분; 변성(denaturation) 95℃ 30초, 결합(annealing) 각 프라이머별 특정 온도(표 1 참고)에서 30초, 신장(extension) 72℃ 30초의 과정을 총 35회 반복; 최종 신장 72℃ 5분의 조건으로 수행하였으며, PCR 증폭 산물은 5300 Fragment analyzer(agilent)로 전기영동하여 확인하였고, 결과 데이터 수집은 Prosize 3.0으로 하였다.The PCR process was pre-denaturation at 95° C. for 15 minutes; The process of denaturation at 95°C for 30 seconds, annealing at a specific temperature for each primer (see Table 1) for 30 seconds, and extension at 72°C for 30 seconds was repeated a total of 35 times; The final elongation was performed under the conditions of 5 minutes at 72°C, and PCR amplification products were confirmed by electrophoresis with a 5300 Fragment analyzer (agilent), and the resulting data were collected using Prosize 3.0.
실시예 1. LMO 유채 계통 검정용 양성대조군 클론의 검증Example 1. Verification of positive control clones for LMO rapeseed line assay
본 발명의 양성대조군 클론은 Ms8, Rf3, GT73(RT73), T45, Ms1, Rf1, Rf2, Topas19-2, Mon88302, DP-073496-4, Ms11, LBFLFK 및 NS-B50027-4 유전자변형 유채 계통 검사용 및 내재 유전자 CruA 확인용으로 구성되어 있다. The positive control clones of the present invention are Ms8, Rf3, GT73 (RT73), T45, Ms1, Rf1, Rf2, Topas19-2, Mon88302, DP-073496-4, Ms11, LBFLFK and NS-B50027-4 genetically modified rapeseed lineage test and for identification of the endogenous gene CruA .
상기 유채 양성대조군 클론의 효율성을 확인하기 위해서 10 ng, 5 ng, 2.5 ng, 1.25 ng, 0.625 ng, 0.3125 ng, 0.15625 ng, 0.078125 ng 농도의 양성대조군 클론을 주형으로 이용하여 PCR을 수행한 결과, 유채 양성대조군 클론 제작에 사용된 프라이머 세트들이 8개 농도에서 모두 예상되는 크기의 PCR 산물을 증폭하는 것을 확인하였다(도 1 내지 도 4). 이를 통해, 본 발명의 양성대조군 클론은 10 ng에서 0.07 ng까지 높은 검출 효율성을 보이고, ng 당 높은 카피수를 가지는 특징이 있음을 알 수 있었다.In order to confirm the efficiency of the rapeseed positive control clone, PCR was performed using positive control clones at concentrations of 10 ng, 5 ng, 2.5 ng, 1.25 ng, 0.625 ng, 0.3125 ng, 0.15625 ng, and 0.078125 ng as a template, It was confirmed that the primer sets used in the production of rapeseed positive control clones amplified PCR products of the expected size at all 8 concentrations (FIGS. 1 to 4). Through this, it was found that the positive control clone of the present invention showed high detection efficiency from 10 ng to 0.07 ng and had a high copy number per ng.
기존에 유전자변형 유채 식물체를 검정하기 위해서는 검정하고자 하는 유채 계통과 내재 유전자를 주형으로 하여 각각 PCR을 수행한 후 증폭된 산물을 정제하고 핵산을 수득하는 과정을 통해 (양성)대조군을 제작하였는데, 기존의 방식으로 제조된 대조군은 검사시료 유래 증폭산물과 동일한 염기서열로 이루어져 있어 대조군의 실험실 오염 시에 구분이 불가하고, 각각의 대조군 핵산을 정제해야 하는 번거로움과 반복 사용으로 인해 대조군의 오염에 대한 문제가 있었다.Previously, in order to test genetically modified rapeseed plants, PCR was performed using the rapeseed strain and the endogenous gene to be tested as a template, and then a (positive) control was prepared through the process of purifying the amplified product and obtaining nucleic acid. The control prepared by the method of is composed of the same nucleotide sequence as the test sample-derived amplification product, so it is impossible to distinguish the control from contamination in the laboratory, and the hassle of purifying each control nucleic acid and repeated use prevent contamination of the control. There was a problem.
반면에, 본 발명의 양성대조군 클론은 LMO 유채 계통별 및 유채 내재 유전자에 특이적인 프라이머 세트 16개(표 1)의 염기서열 이외에 LMO 유채와 관련이 없는 인위적인 서열로 구성되어 있어 양성대조군의 오염 확인이 용이하며, 다양한 LMO 유채 계통 및 내재 유전자에 특이적인 프라이머 세트가 모두 포함되어 있음에도 불구하고 비특이적 반응없이 16개의 프라이머 세트들이 각각 모두 예상되는 크기의 PCR 산물을 증폭하는 것이 가능하므로, 본 발명의 양성대조군 클론은 기존의 양성대조군에 비해 LMO 유채 식물체를 보다 정확하고 효율적으로 검정할 수 있는 양성대조군임을 알 수 있었다.On the other hand, the positive control clone of the present invention consists of an artificial sequence unrelated to LMO rapeseed, in addition to the nucleotide sequence of 16 primer sets (Table 1) specific for each LMO rapeseed line and rapeseed endogenous gene, confirming contamination of the positive control. This is easy, and despite the fact that all primer sets specific to various LMO rapeseed strains and endogenous genes are included, it is possible to amplify PCR products of the expected size with each of the 16 primer sets without non-specific reaction, so the positive result of the present invention It was found that the control clone is a positive control that can more accurately and efficiently test LMO rapeseed plants compared to the existing positive control.
따라서, 본 발명의 양성대조군 클론은 표 1에 개시된 LMO 유채 계통별 및 내재 유전자 특이적 프라이머 세트에 대한 양성대조군으로 사용할 수 있으므로, 기존 PCR 검사법을 이용하여 다양한 유전자변형 유채를 검정하는데 유용하게 활용될 수 있을 것이다.Therefore, since the positive control clones of the present invention can be used as positive controls for the LMO rapeseed strains and endogenous gene-specific primer sets disclosed in Table 1, they can be usefully used to test various genetically modified rapeseeds using conventional PCR testing methods. You will be able to.
<110> REPUBLIC OF KOREA(Animal and Plant Quarantine Agency) <120> Positive control plasmid for screening genetically modified canola and method for screening genetically modified canola using the same <130> PN22008 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 972 <212> DNA <213> Artificial Sequence <220> <223> positive control plasmid <400> 1 aaccatattg accatcatac tcattgctga ctaaccacgt aaagaccaac aggcaccaaa 60 tcctggccag ggtttccgtg attccttcct tttcttgcct tcgtataagc tcgtggatat 120 acaacacgtg actgtaaagg atatccaatg gttctacaac gacggaagag catttagcat 180 gtaccatcag acagattaac gtggaactga cagaaccgca acaattgcga cgtacggcta 240 agagcgaatt tggcactaac ccttgaggaa actggtagca catctcaagt ctccatcttc 300 ctttatgatt ccttatctgg gaactactca cgcgttactt acctctctga cttttccacc 360 aaggaacgtt ggggaaatta aattagctac atcttgacct cccttagggc ccaattccgg 420 atcggtcgtc gatatattgt ctcacccctc cattgagcag tgaacaccaa gttgttagaa 480 aaagtaaaca attaatatag ccggaagatg ggaattaaca tctacaaatt gcagtgttgt 540 ggtggtgacg atttacaggc caaattcgct cttagccgta gtgataacca aaaacaccct 600 ccaaccctgc tgctttacat ggatttctcc cgtccatatt gaccatcata ctcattgcga 660 catttggaaa ccttcattag agtgttcaaa cactgatagt ttaaactgaa tgtaaacgac 720 aatctgctag tggaccaaag cgagatgaat aattcctttt cttatcctgt tgaccaaacc 780 atcatactca ttgctgatcc aaggtccgtg ggccttccta aacgtgccgt aatttacccg 840 aagattggta atatgtaaat aacgggatcc aggggaggta tactatgtgt gcgtatacag 900 gaactatgga ggtttgtatg aactgatgat ctaggaccgg ataaccggga ggtctacagg 960 ccaaattcgc aa 972 <110> REPUBLIC OF KOREA (Animal and Plant Quarantine Agency) <120> Positive control plasmid for screening genetically modified canola and method for screening genetically modified canola using the same <130> PN22008 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 972 <212> DNA <213> artificial sequence <220> <223> positive control plasmid <400> 1 aaccatattg accatcatac tcattgctga ctaaccacgt aaagaccaac aggcaccaaa 60 tcctggccag ggtttccgtg attccttcct tttcttgcct tcgtataagc tcgtggatat 120 acaacacgtg actgtaaagg atatccaatg gttctacaac gacggaagag catttagcat 180 gtaccatcag acagattaac gtggaactga cagaaccgca acaattgcga cgtacggcta 240 agagcgaatt tggcactaac ccttgaggaa actggtagca catctcaagt ctccatcttc 300 ctttatgatt ccttatctgg gaactactca cgcgttactt acctctctga cttttccacc 360 aaggaacgtt ggggaaatta aattagctac atcttgacct cccttagggc ccaattccgg 420 atcggtcgtc gatatattgt ctcacccctc cattgagcag tgaacaccaa gttgttagaa 480 aaagtaaaca attaatatag ccggaagatg ggaattaaca tctacaaatt gcagtgttgt 540 ggtggtgacg atttacaggc caaattcgct cttagccgta gtgataacca aaaacaccct 600 ccaaccctgc tgctttacat ggatttctcc cgtccatatt gaccatcata ctcattgcga 660 catttggaaa ccttcattag agtgttcaaa cactgatagt ttaaactgaa tgtaaacgac 720 aatctgctag tggaccaaag cgagatgaat aattcctttt cttatcctgt tgaccaaacc 780 atcatactca ttgctgatcc aaggtccgtg ggccttccta aacgtgccgt aatttacccg 840 aagattggta atatgtaaat aacgggatcc agggggaggta tactatgtgt gcgtatacag 900 gaactatgga ggtttgtatg aactgatgat ctaggaccgg ataaccggga ggtctacagg 960 ccaaattcgc aa 972
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