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PCR Detection of Disease Resistant Transgenic Rice for the Biosafety Assessment
Kong Sik Shin,Myung Ho Lim,Hee Jong Woo,Jin Hyoung Lee,Hong Il Ahn,Yang Qin,Hyun Suk Cho 한국육종학회 2012 한국육종학회 심포지엄 Vol.2012 No.07
Regulations in the EU, Japan, Korea, etc. require that foods and feeds made of or derived from genetically modified organisms (GMOs) should be approved and labeled according to a threshold. Recently, disease resistant transgenic rice was developed in Korea, which resulted from the transformation events involving choline kinase gene, OsCK1. In order to monitor unintended release of the developed GM rice in the near future, as well as to meet GM-labeling requirements, the development of a reliable method for detection of disease resistant GM rice is requisite. Here, specific primer pairs for the detection of GMO was designed on the basis of a introduced gene and the flanking junction sequences between a plant DNA and a integrated gene construct, and also SPS gene was used as an endogenous reference material. Specificities of all designed primers were tested through qualitative PCRs. Clearly, target specific amplicons could be detected from disease resistant GM rice event. In addition, the limits of detection (LOD) using the event-specific primers were approximately 0.1% for the disease resistant GM rice line. This result indicated that the developed detection method is suitable for the traceability of disease resistant GM rice, because of using the primer specifically corresponded to the junction site between plant genomic DNA and inserted DNA. Keywords: genetically modified organisms, disease resistant GM rice, PCR detection, event-specific primer
Kong-Sik Shin,Myung-Ho Lim,Hee-Jong Woo,Sun-Hyung Lim,Hong-Il Ahn,Jin-Hyoung Lee,Hyun-Suk Cho,Soon-Jong Kweon,Seok Cheol Suh 한국응용생명화학회 2012 Applied Biological Chemistry (Appl Biol Chem) Vol.55 No.3
Transgenic Chinese cabbage 416-3 was developed in Korea by a transformation event involving modified insectresistant gene (cry1Ac1). To monitor unintended release of genetically modified (GM) Chinese cabbage in the future, as well as to meet GM-labeling requirements, the development of a reliable method for detection of GM cabbage is requisite. To develop qualitative and quantitative polymerase chain reaction methods for the insect-resistant GM Chinese cabbage, a cytosolic glutathione reductase (BcgGR1) gene was used as the endogenous reference gene. Primer pairs CGR-1/-2, amplifying the Chinese cabbage endogenous gene, yielded an expected amplicon of 121bp, whereas no amplified product was observed when DNA samples from seven non-cabbage plants were used as templates. The event-specific primer pairs amplifying the junction site between the endogenous genome sequence and the transferred DNA of GM event 416-3, produced amplicons of desired size by qualitative polymerase chain reaction (PCR) assay. An eventspecific quantitative PCR detection method was established using a TaqMan probe and a standard plasmid as a reference molecule,which contained both endogenous and event-specific sequences. For the validation of this method, three different compositions of w/w mixed samples (containing transgenic DNA at 5, 1, and 0.5%of total DNA in the control samples) were quantified. The precision, expressed as standard deviation (SD) and relative standard deviations (RSD), deviated by 0.03–0.26% and 4.75–8.06%, respectively. These results clearly demonstrate that the PCR methods developed herein can be used for event-specific qualitative and quantitative testings of insect-resistant GM Chinese cabbage.
Shin, Kong-Sik,Lim, Myung-Ho,Woo, Hee-Jong,Lim, Sun-Hyung,Ahn, Hong-Il,Lee, Jin-Hyoung,Cho, Hyun-Suk,Kweon, Soon-Jong,Suh, Seok-Cheol The Korean Society for Applied Biological Chemisty 2012 Journal of Applied Biological Chemistry (J. Appl. Vol.58 No.3
Transgenic Chinese cabbage 416-3 was developed in Korea by a transformation event involving modified insect-resistant gene (cry1Ac1). To monitor unintended release of genetically modified (GM) Chinese cabbage in the future, as well as to meet GM-labeling requirements, the development of a reliable method for detection of GM cabbage is requisite. To develop qualitative and quantitative polymerase chain reaction methods for the insect-resistant GM Chinese cabbage, a cytosolic glutathione reductase (BcgGR1) gene was used as the endogenous reference gene. Primer pairs CGR-1/-2, amplifying the Chinese cabbage endogenous gene, yielded an expected amplicon of 121 bp, whereas no amplified product was observed when DNA samples from seven non-cabbage plants were used as templates. The event-specific primer pairs amplifying the junction site between the endogenous genome sequence and the transferred DNA of GM event 416-3, produced amplicons of desired size by qualitative polymerase chain reaction (PCR) assay. An event-specific quantitative PCR detection method was established using a TaqMan probe and a standard plasmid as a reference molecule, which contained both endogenous and event-specific sequences. For the validation of this method, three different compositions of w/w mixed samples (containing transgenic DNA at 5, 1, and 0.5% of total DNA in the control samples) were quantified. The precision, expressed as standard deviation (SD) and relative standard deviations (RSD), deviated by 0.03-0.26% and 4.75-8.06%, respectively. These results clearly demonstrate that the PCR methods developed herein can be used for event-specific qualitative and quantitative testings of insect-resistant GM Chinese cabbage.
연구보문 : 알파토코페놀 고 함유 유전자변형 들깨에 대한 검정법 개발
신공식 ( Kong Sik Shin ),우희종 ( Hee Jong Woo ),이기종 ( Ki Jong Lee ),김경환 ( Kyung Hwan Kim ),권순종 ( Soon Jong Kweon ),서석철 ( Seok Cheol Suh ) 한국국제농업개발학회 2010 韓國國際農業開發學會誌 Vol.22 No.4
알파토코페놀을 증가시키기 위해서 γ-TMT(γ-tocopherol methyltransferase)유전자로 형질전환 된 유전자변형 들깨가 개발되었고, 도입유전자의 검출법 개발을 위하여 정성 및 정량 PCR 분석을 수행하였다. 도입유전자 및 들깨 내재유전자를 바탕으로 하여 몇 개의 검출 primer쌍을 제조하였으며, 정성 PCR 방법으로 primer의 특이성을 조사하였다. 들깨 내재 유전자, KAS-I의 증폭을 위해서 KASI02-1/2 primer를 사용하여 195 bp 크기의 PCR 증폭산물을 얻었으며, 도입유전자에 대하여 구조 특이적 primer, TMTocs-1/2 및 VicTM-1/2를 이용하여 유전자변형들깨를 포함한 6개 작물과 국내 개발된 3개 유전자변형 작물에 대해 PCR을 수행한 결과에서 각각 191 bp 및 109 bp의 PCR 산물이 유전자변형 들깨에서만 특이적으로 증폭되는 것을 확인하였다. 정량 검출을 위해서 plasmid, pKAViTM을 제조하였고, 이를 표준시료로 하여, 0.3, 1 및 1.5%로 조제된 유전자변형 들깨를 real-time PCR로 분석함으로써 유의성 있는 결과를 얻을 수 있었으며, 이의 방법이 유전자변형 들깨를 정량적으로 검정하는데 적용될 수 있음을 확인하였다. Qualitative and quantitative PCR methods were performed to examine the detection of γ-TMT(γ-tocopherol methyltransferase) inserted into genetically modified(GM) perilla(Perilla frutescens)developed in Korea. Several primer pairs were prepared on introduced genes and an endogenous reference gene in perilla. Specificity of primers first was tested by the means of qualitative PCR analysis. Primer pair KASI02-1/2 was used to amplify the endogenous gene, KAS-I, and gave rise to an amplicon 195 bp. PCR amplification using the construct-specific primer pairs, TMTocs-1/2 and VicTM-1/2 also was performed for GM perilla. TMTocs-1/2 and VicTM-1/2 primers gave rise to an amplicon 191 bp and 109 bp, respectively. In contrast, no amplified product was observed when DNA samples from 6 different plants and 3 GM crops were used as templates. For quantitative detection, test samples containing 0.3, 1, and 1.5% genetically modified perilla were measured by real-time PCR using the plasmid DNA, pKAViTM as standard material. This result showed real-time PCR method was applicable to detect GM perilla quantitatively.