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국내 승인 LM면화의 자연환경 모니터링을 위한 multiplex PCR 개발
조범호,설민아,신수영,김일룡,최원균,엄순재,송해룡,이중로,Jo, Beom-Ho,Seol, Min-A,Shin, Su Young,Kim, Il Ryong,Choi, Wonkyun,Eum, Soon-Jae,Song, Hae-Ryong,Lee, Jung Ro 한국식물생명공학회 2016 식물생명공학회지 Vol.43 No.1
면화(cotton)는 섬유를 수확하고, 면실유는 식용으로 가공 후 남은 것은 다시 사료로 이용되어 다방면으로 다양하게 활용되는 작물이다. LM 면화는 옥수수, 대두, 캐놀라와 달리 아시아권(중국, 인도 등)에서 가장 많이 재배되고 있으며, 우리나라는 2013년까지 세계 1위의 LM 면실(사료용) 수입국이며, 세계 4위의 면실박 수입국으로 해마다 증가하는 LM 면화의 수입량과 맞물려 유통 및 소비과정에서의 비의도적으로 유출 가능성이 증가함에 따라 LM 면화의 자연생태계 위해성 평가 및 안전관리가 요구된다. 본 연구에서는 국내 수입 승인 LM 면화 6개 이벤트(MON15985, MON531, GHB614, LLCOTTON25, MON88913, MON1445)의 동시증폭 검출법(multiplex PCR)을 개발하여 보다 신속하고 명확한 검출기법을 확립하고자 하였다. 최적 multiplex PCR 반응 조건은 2개 LM 면화 이벤트 MON15985 (214 bp), MON531 (270 bp)와 4개 LM 면화 이벤트 GHB614 (119 bp), LLCOTTON25 (164 bp), MON88913 (276 bp), MON1445 (389 bp)가 한번의 반응에 명확하게 검출되는 최적 반응 조건 및 primer 반응 농도의 조절을 통해 서로 다른 생성물 크기로 명확히 구분되도록 하였고, 최적 primer 농도는 반응액 최종농도 0.2~0.66 pmol로 primer 쌍 마다 각각 다른 최적 농도 조합의 cocktail을 만들어 활용하였다. Duplex PCR 반응 조건은 초기 $95^{\circ}C$ 5분 반응 후, $95^{\circ}C$ 15초, $55^{\circ}C$ 20초 15회 반응하고, 다시 $95^{\circ}C$ 15초, $60^{\circ}C$ 20초 25회 반응하였을 때 최적 검출이 이루어졌고, tetraplex PCR 반응 조건은 $95^{\circ}C$ 5분 반응 후, $95^{\circ}C$ 15초, $60^{\circ}C$ 20초 50회 반응하였을 때 최적 검출이 이루어졌다. 본 연구에서 개발된 multiplex PCR 검출법은 국내 수입 유통 LM 면화의 자연환경 모니터링에 활용함에 있어 요구되는 연구인력, 시간 및 비용을 보다 효율적으로 개선하는데 적용될 수 있을 것으로 사료된다. The growth area of living modified (LM) cotton has steadily increased every year, since its first commercialization in 1996. Development of environmental risk assessment tools and techniques for LM cotton is required for ecosystem safety. We therefore developed multiplex PCR assays for simultaneous detection of two (MON15985, MON531) and four (GHB614, LLCOTTON25, MON88913 and MON1445) LM cotton events approved in Korea, with event specific primer pairs. The PCR reactions were optimized by using event specific primers of six LM cottons at various concentrations. The reactions allows amplification of estimated amplicons of MON15985 (214 bp), MON531 (270 bp), GHB614 (119 bp), LLCOTTON25 (164 bp), MON88913 (276 bp), and MON1445 (389 bp) from multiplex PCR reactions. The multiplex PCR assay developed allowed that two annealing steps (15 cycles at $55^{\circ}C$ and 25 cycles at $60^{\circ}C$) were performed for amplification of distinguished two LM cottons, and only one annealing step (50 cycles at $60^{\circ}C$) was necessary for tetraplex PCR. Primer extension step of all PCR reactions was skipped for time-effective amplification. Our methods suggest that two multiplex PCR assays can be cost-effective and a rapid diagnostic tool for environmental LMO monitoring of six LM cottons.
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Characterization of Polymorphic Microsatellite Loci in Cultivated and Wild Panax ginseng
조범호,서동상,조은민,김준기,류기형,정기화 한국유전학회 2009 Genes & Genomics Vol.31 No.2
Ginseng ( Panax ginseng) is one of the most important herbal remedies used in East Asia. The present study investigated six polymorphic microsatellite markers (PG29, PG281, PG287, PG668, PG1319, and PG1481) in samples of cultivated and wild P. ginseng collected in Korea. Total allelic number observed in this study was 27 (average allelic numbers per locus: 4.5). All examined loci exhibited deviation from the Hardy-Weinberg equilibrium and deficiency of heterozygosity in both cultivated and wild groups. Although the wild ginseng group exhibited slightly more polymorphic behavior (mean PIC = 0.392, GD= 0.454 and Hobs = 0.129), compared with the cultivated group (mean PIC = 0.383, GD= 0.438 and Hobs = 0.105), no significant differences of allele frequencies and genotype distributions were revealed. By combined analysis of six loci in 100 cultivated ginsengs, 71 different types were observed. The analyzed microsatellite loci in this study will be helpful for understanding genetic variation, QTL mapping and phylogenic studies in Panax species. Ginseng ( Panax ginseng) is one of the most important herbal remedies used in East Asia. The present study investigated six polymorphic microsatellite markers (PG29, PG281, PG287, PG668, PG1319, and PG1481) in samples of cultivated and wild P. ginseng collected in Korea. Total allelic number observed in this study was 27 (average allelic numbers per locus: 4.5). All examined loci exhibited deviation from the Hardy-Weinberg equilibrium and deficiency of heterozygosity in both cultivated and wild groups. Although the wild ginseng group exhibited slightly more polymorphic behavior (mean PIC = 0.392, GD= 0.454 and Hobs = 0.129), compared with the cultivated group (mean PIC = 0.383, GD= 0.438 and Hobs = 0.105), no significant differences of allele frequencies and genotype distributions were revealed. By combined analysis of six loci in 100 cultivated ginsengs, 71 different types were observed. The analyzed microsatellite loci in this study will be helpful for understanding genetic variation, QTL mapping and phylogenic studies in Panax species.
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Multiplex PCR 방법을 이용한 국내 승인 5개 LM 유채의 검출법 개발
조범호,이중로,최원균,문정찬,신수영,엄순재,설민아,김일룡,송해룡 한국식물생명공학회 2015 식물생명공학회지 Vol.42 No.2
Canola is a crop globally used for production of oil and biofuel. Cultivation area and import volume of living modified (LM) canola have been increasing every year. As canola import dependence has reached 100% in Korea, efforts have been made for safety management of LM canola and ecological risk assessment. We developed a set of multiplex PCR method for simultaneous detection of 5 LM canola events (Topas 19/2, Rf3, Ms8, RT73 and T45) approved in Korea. The multiplex PCR assay developed allows amplification of estimated products of 5 LM canolas from event specific primer sets. Primer extension time was skipped for a time-consuming process and two annealing steps (20 cycles at 55°C and 20 cycles at 60°C) were performed for yielding the best result which was sufficient to distinguish five LM canolas. Our results suggest that multiplex PCR method provides a cost and time-effective approach for LM canola detection.
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국내 승인 LM면화의 자연환경 모니터링을 위한 multiplex PCR 개발
조범호,설민아,신수영,김일룡,최원균,엄순재,송해룡,이중로 한국식물생명공학회 2016 JOURNAL OF PLANT BIOTECHNOLOGY Vol.43 No.1
The growth area of living modified (LM) cotton has steadily increased every year, since its first commercialization in 1996. Development of environmental risk assessment tools and techniques for LM cotton is required for ecosystem safety. We therefore developed multiplex PCR assays for simultaneous detection of two (MON15985, MON531) and four (GHB614, LLCOTTON25, MON88913 and MON1445) LM cotton events approved in Korea, with event specific primer pairs. The PCR reactions were optimized by using event specific primers of six LM cottons at various concentrations. The reactions allows amplification of estimated amplicons of MON15985 (214 bp), MON531 (270 bp), GHB614 (119 bp), LLCOTTON25 (164 bp), MON88913 (276 bp), and MON1445 (389 bp) from multiplex PCR reactions. The multiplex PCR assay developed allowed that two annealing steps (15 cycles at 55°C and 25 cycles at 60°C) were performed for amplification of distinguished two LM cottons, and only one annealing step (50 cycles at 60°C) was necessary for tetraplex PCR. Primer extension step of all PCR reactions was skipped for time-effective amplification. Our methods suggest that two multiplex PCR assays can be cost-effective and a rapid diagnostic tool for environmental LMO monitoring of six LM cottons. 면화(cotton)는 섬유를 수확하고, 면실유는 식용으로 가공후 남은 것은 다시 사료로 이용되어 다방면으로 다양하게 활용되는 작물이다. LM 면화는 옥수수, 대두, 캐놀라와 달리 아시아권(중국, 인도 등)에서 가장 많이 재배되고 있으며, 우리나라는 2013년까지 세계 1위의 LM 면실(사료용) 수입국이며, 세계 4위의 면실박 수입국으로 해마다 증가하는 LM 면화의 수입량과 맞물려 유통 및 소비과정에서의 비의도적으로 유출 가능성이 증가함에 따라 LM 면화의 자연생태계 위해성 평가 및 안전관리가요구된다. 본 연구에서는 국내 수입· 승인 LM 면화 6개이벤트(MON15985, MON531, GHB614, LLCOTTON25, MON 88913, MON1445)의 동시증폭 검출법(multiplex PCR)을 개발하여 보다 신속하고 명확한 검출기법을 확립하고자 하였다. 최적 multiplex PCR 반응 조건은 2개 LM 면화 이벤트 MON15985 (214 bp), MON531 (270 bp)와 4개 LM 면화이벤트 GHB614 (119 bp), LLCOTTON25 (164 bp), MON 88913 (276 bp), MON1445 (389 bp)가 한번의 반응에 명확하게 검출되는 최적 반응 조건 및 primer 반응 농도의 조절을 통해 서로 다른 생성물 크기로 명확히 구분되도록하였고, 최적 primer 농도는 반응액 최종농도 0.2 ~ 0.66 pmol로 primer 쌍 마다 각각 다른 최적 농도 조합의cocktail 을 만들어 활용하였다. Duplex PCR 반응 조건은 초기 95°C 5분 반응 후, 95°C 15초, 55°C 20초 15회 반응하고, 다시95°C 15초, 60°C 20초 25회 반응하였을 때 최적 검출이 이루어졌고, tetraplex PCR 반응 조건은 95°C 5분 반응 후, 95°C 15초, 60°C 20초 50회 반응하였을 때 최적 검출이 이루어졌다. 본 연구에서 개발된 multiplex PCR 검출법은 국내 수입·유통 LM 면화의 자연환경 모니터링에 활용함에있어 요구되는 연구인력, 시간 및 비용을 보다 효율적으로 개선하는데 적용될 수 있을 것으로 사료된다.
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벤토나이트 슬러리(Bentonite Slurry)의 물리적(物理的) 특성(特性)에 관(關)한 연구(硏究)
조범호 ( Bum Ho Cho ),유기송 ( Ki Song Ryu ) 한국농공학회 1983 韓國農工學會誌 : 전원과 자원 Vol.25 No.1
The study was conducted to find out the physical properties of bentonite slurry. For this study, high and low quality-bentonite from the area near Yeongil Bay, Gyeong-buk Province, were used The results of study are summarized as follows: 1. The specific gravity of slurry is increased in proportion to the bentonite contents, and the correlation between the specific gravity of slurry (d) and bentonite contents (B) is presented as d=0.005B+0.003. 2. The viscosity of slurry increase in accordance with the increments of bentonite contents and time elapsed, and the increment rate of viscosity of high-quality- bentonite is more prominent than that of low-quality-bentonite. 3. The thickness of filter cake and infiltration loss decrease in proportion to the increments of bentonite slurry viscosity, but when the viscosity of bentonite slurry is beyond 30sec-Marsh, both of them are almost constant. 4. The pH of slurry decrease in proportion to the increment of time elapsed and bentonite contents. 5. The test results showed that the viscosity of bentonite slurry should be bigger than 30 sec-Marsh for the stability of the slurry trench.
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